Methods and kits for classifying cannabinoid production in cannabis plants

ABSTRACT

The present disclosure provides methods and kits for characterizing  Cannabis  plants. Methods and kits of the present disclosure include detection/amplification of one or more enzymes involved in the production of cannabinoids, such as, for example, tetrahydrocannabinolic acid (THCA) synthase and/or cannabidiolic acid (CBDA) synthase.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority of U.S. ProvisionalApplication No. 62/598,967, filed Dec. 14, 2017, all of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 5, 2019, isnamed Corrected Sequence Listing 2010807-0026.tx and 31 KB in size.

BACKGROUND

C. sativa is an annual herbaceous flowering plant that has beencultivated throughout recorded history for use as fiber, seed oil, food,medicine and recreation. Cannabinoids are secondary metabolites found inC. sativa plants that can act on cannabinoid receptors in cells thatalter neurotransmitter release in the brain. More than 100 cannabinoidshave been isolated from C. sativa, which have varied effects on mammalsto which they have been administered.

SUMMARY

The present disclosure encompasses the recognition that certaingenotypes are associated with altered cannabinoid synthesis or analtered cannabinoid profile. For example, the present disclosurerecognizes that certain variants (e.g., wild-type and/or mutated) oftetrahydrocannabinolic acid (THCA) synthase gene sequences and/orcannabidiolic acid (CBDA) synthase gene sequences can provide insightregarding synthesis of tetrahydrocannabinol (THC) and/or cannabidiol(CBD) in a Cannabis plant. The present disclosure provides aclassification system for Cannabis, which designates a Cannabis plant asbeing of a particular type based on its genotype, which includes theclassifications illustrated in FIG. 2 and outlined in Table 1 below:

TABLE 1 CBDA Genotype THCA Genotype Classification CBDA⁻/CBDA⁻THCA⁺/THCA⁺ Type Ia Plant CBDA⁻/CBDA⁻ THCA⁺/THCA⁻ Type Ib PlantCBDA⁺/CBDA⁻ THCA⁺/THCA⁺ Type IIa Plant CBDA⁺/CBDA⁻ THCA⁺/THCA⁻ Type IIbPlant CBDA⁺/CBDA⁺ THCA⁺/THCA⁺ Type IIb Plant CBDA⁺/CBDA⁺ THCA⁺/THCA⁻Type IIc Plant CBDA⁺/CBDA⁻ THCA⁻/THCA⁻ Type IIIa Plant CBDA⁺/CBDA⁺THCA⁻/THCA⁻ Type IIIb Plant CBDA⁻/CBDA⁻ THCA⁻/THCA⁻ Type IV Plant

Such a classification system can be utilized, e.g., in the selection ofa Cannabis plant for various purposes. For example, a Type IIIb Cannabisplant (CBDA⁺/CBDA⁺ and THCA⁻/THCA⁻) may be useful as a therapeutic,particularly in applications in which psychoactive effects caused by THCare undesirable. The present disclosure also recognizes Type V Cannabisplants. In some embodiments, a Type V Cannabis plant is characterized bya mutation in one or both of a olivetol synthase gene sequence and adivarinic acid synthase gene sequence. Olivetolic acid and divarinicacid are produced by enzymes olivetol synthase and divarinic acidsynthase, respectively, in the cannabinoid synthesis pathway (see, e.g.,FIG. 1, panel (A)).

The present disclosure further recognizes certain limits associated withprevious assays to characterize THCA synthase, e.g., an inability todetect a THCA synthase gene sequence (e.g., because assays use primersor probes are complementary to highly variant sequences), and/or afalse-positive detection (e.g., by amplification of a THCA synthasepseudogene). The present disclosure provides a method that reliably andaccurately detects a THCA synthase gene sequence in a Cannabis plant.The present disclosure provides the insight that detection of a THCAsynthase gene sequence (e.g., a wild-type or mutated THCA synthase genesequence) present in a Cannabis plant can be achieved by amplificationof a THCA synthase gene sequence using one or more primers withsequences complementary to sequences (e.g., promoter) upstream of thecoding sequence and (e.g., terminator region, 5′ UTR, etc.) downstreamof the coding sequence may be particularly useful for in vitro detectionof a functional THCA synthase gene sequence. Moreover, the presentdisclosure provides methods that include detection of a wild-type THCAsynthase gene sequence and a mutant version of a THCA synthase genesequence, thereby indicating the THCA synthase genotype of the plant.

The present disclosure also provides a method that reliably andaccurately detects a CBDA synthase gene sequence in a Cannabis plant.The present disclosure provides methods that include detection of awild-type CBDA synthase gene sequence and a mutant version of a CBDAsynthase gene sequence, thereby indicating the CBDA synthase genotype ofthe plant. In some embodiments, detection of a wild-type CBDA synthasegene sequence in a Cannabis plant can be achieved by amplification of aCBDA synthase gene sequence using a primer that is complementary (atleast in part) to a sequence of a first CBDA synthase gene sequence(e.g., CGTA at residues 330-333 in FIG. 4). In some embodiments,detection of a mutant CBDA synthase gene sequence in a Cannabis plantcan be achieved by amplification of a CBDA synthase gene sequence usinga primer that is complementary (at least in part) to a CBDA synthasegene sequence that includes a deletion of the first sequence (e.g.,ACTTAC at residues 327-329 and 334-336 in FIG. 4).

In some aspects, the present disclosure relates to methods and kits forclassifying cannabinoid production in a Cannabis plant, such as a C.sativa plant, a C. indica plant, or a C. ruderalis plant.

In some aspects, the present disclosure provides methods that includedetection of one or more nucleic acid sequences in a sample of aCannabis plant. In some embodiments, a method includes detecting thepresence of a tetrahydrocannabinolic acid (THCA) synthase gene sequence,e.g., a THCA synthase gene sequence comprising a THCA synthase codingregion (e.g., an entire THCA synthase coding region) and/or a THCAsynthase gene sequence promoter. In some embodiments, a method includesdetecting the presence of a mutated THCA synthase gene sequence. In someembodiments, a mutated THCA synthase gene sequence includes at least onemutation that alters the activity of a THCA synthase encoded by themutated THCA synthase gene sequence relative to a THCA synthase encodedby a wild-type THCA synthase gene sequence. In some embodiments, amethod includes detecting the presence of a wild-type cannabidiolic acid(CBDA^(wt)) synthase gene sequence. In some embodiments, a methodincludes detecting the presence of a mutated cannabidiolic acid(CBDA^(mut)) synthase gene sequence. In some embodiments, a mutated CBDAsynthase gene sequence comprises a deletion mutation.

In some embodiments, a method includes detecting a wild-type THCAsynthase gene sequence, a mutant THCA synthase gene sequence, awild-type CBDA synthase gene sequence, a mutant CBDA synthase genesequence, or any combination thereof from a sample that comprisesCannabis nucleic acid (i.e., nucleic acid of a Cannabis plant). In someembodiments, a method further includes detecting one, two, three, four,or more of a wild-type olivetol synthase gene sequence, a variantolivetol synthase gene sequence, a wild-type divarinic acid synthasegene sequence, a variant divarinic acid synthase gene sequence, awild-type limonene synthase gene sequence, and a variant limonenesynthase gene sequence from a sample that comprises Cannabis nucleicacid.

In some embodiments, detection of one or more nucleic acid sequences ina sample of Cannabis plant includes isothermic nucleic acidamplification. In some embodiments, isothermic nucleic acidamplification is a Loop-Mediated Isothermal Amplification (LAMP) assay.In some embodiments, a LAMP assay is a colorimetric LAMP assay.

In some embodiments, detection of a THCA synthase gene sequence includesamplification of a THCA synthase gene sequence that is at least 2000nucleotides long. In some certain embodiments, an amplified THCAsynthase gene sequences is 1500-2500, 1750-2250, 2000-2200, or 2100-2200nucleotides long. In some embodiments, an amplified THCA synthase genesequence includes at least part of the THCA synthase promoter and 5′UTR.

In some embodiments, a Cannabis plant is a C. sativa plant. In someembodiments, a Cannabis plant is a C. indica plant. In some embodiments,a Cannabis plant is a C. ruderalis plant.

In some embodiments, detecting the presence of a THCA synthase genesequence includes contacting a sample that comprises nucleic acid fromthe Cannabis plant with at least two THCA synthase primers underconditions sufficient for amplification of a THCA synthase genesequence. In some certain embodiments, detecting the presence of a THCAsynthase gene sequence includes contacting nucleic acid from a Cannabisplant with at least two primers that are at least 70%, 75%, 80%, 85%,85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to any of SEQ ID NOs: 8-12.

In some certain embodiments, detecting the presence of a THCA synthasegene sequence is by LAMP amplification and includes contacting nucleicacid from a Cannabis plant with four or more primers that are at least70%, 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 8-12.

In some embodiments, detecting the presence of a mutated THCA synthasegene sequence includes contacting a sample that comprises nucleic acidfrom the Cannabis plant with at least two THCA synthase primers underconditions sufficient for amplification of a mutated THCA synthase genesequence.

In some embodiments, detecting the presence of a wild-type cannabidiolicacid (CBDA^(WT)) synthase gene sequence includes contacting a samplethat comprises nucleic acid from the Cannabis plant with at least twoCBDA synthase primers under conditions sufficient for amplification of awild-type CBDA (CBDA^(WT)) synthase gene sequence. In some certainembodiments, detecting the presence of a CBDA^(WT) synthase genesequence includes contacting nucleic acid from a Cannabis plant with atleast two primers that are at least 70%, 75%, 80%, 85%, 85%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto any of SEQ ID NOs: 13-18 and 27.

In some embodiments, detecting the presence of a mutated cannabidiolicacid (CBDA^(mut)) synthase gene sequence includes contacting a samplethat comprises nucleic acid from the Cannabis plant with at least twoCBDA synthase primers under conditions sufficient for amplification of aCBDA^(mut) synthase gene sequence. In some embodiments a CBDA^(mut)synthase is a deletion (CBDA^(del)). In some certain embodiments,detecting the presence of a CBDA^(del) synthase gene sequence includescontacting nucleic acid from a Cannabis plant with at least two primersthat are at least 70%, 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ IDNOs: 13, 15-19, and 27.

In some embodiments, detecting the presence of a THCA synthase genesequence includes contacting a sample that comprises nucleic acid fromthe Cannabis plant with at least five THCA synthase primers underconditions sufficient for LAMP of a THCA synthase gene sequence. In somecertain embodiments, the at least five primer sequences are at least70%, 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to each of SEQ ID NOs: 8-12.

In some embodiments, detecting the presence of a mutated THCA synthasegene sequence includes contacting a sample that comprises nucleic acidfrom the Cannabis plant with at least five THCA synthase primers underconditions sufficient for LAMP of a mutated THCA synthase gene sequence.

In some embodiments, detecting the presence of a wild-type cannabidiolicacid (CBDA^(WT)) synthase gene sequence includes contacting a samplethat comprises nucleic acid from the Cannabis plant with at least fiveCBDA synthase primers under conditions sufficient for LAMP of aCBDA^(WT) synthase gene sequence. In some certain embodiments, detectingthe presence of a CBDA^(WT) synthase gene sequence is by LAMPamplification and includes contacting nucleic acid from a Cannabis plantwith six primers that are at least 70%, 75%, 80%, 85%, 85%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto each of SEQ ID NOs: 13-18 and 27.

In some embodiments, detecting the presence of a mutated cannabidiolicacid (CBDA^(mut)) synthase gene sequence includes contacting a samplethat comprises nucleic acid from the Cannabis plant with at least fiveCBDA synthase primers under conditions sufficient for LAMP of aCBDA^(mut) synthase gene sequence. In some embodiments a CBDA^(mut)synthase is a deletion (CBDA^(del)). In some certain embodiments,detecting the presence of a CBDA^(del) synthase gene sequence is by LAMPamplification and includes contacting nucleic acid from a Cannabis plantwith six primers that are at least 70%, 75%, 80%, 85%, 85%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto each of SEQ ID NOs: 13, 15-19, and 27.

In some embodiments, a method of the present disclosure includes a stepof analyzing results from one or more detecting steps, therebycharacterizing a Cannabis plant as a Type Ia, Type Ib, Type IIa, TypeIIb, Type IIc, Type IIIa, Type IIIb, Type IV or Type V plant.

In some aspects, the present disclosure provides methods that includecontacting a sample that comprises nucleic acid from a Cannabis plantwith at least two primers sufficient for amplification of a Cannabisenzyme gene sequence. In some embodiments, a method includes contactinga sample that comprises nucleic acid from a Cannabis plant with at leasttwo primers sufficient for amplification of a THCA synthase genesequence under conditions sufficient for amplification of a THCAsynthase gene sequence, where at least two THCA synthase primers includea forward THCA synthase primer and a reverse THCA synthase primer. Insome embodiments, a forward THCA synthase primer is complementary to asequence that is 200-1000 nucleotides upstream of a THCA synthase openreading frame in a Cannabis genome. In some embodiments, a reverse THCAsynthase primer is complementary to a sequence that is 50-1000nucleotides downstream of the THCA synthase open reading frame.

In some embodiments, a method includes contacting a sample thatcomprises nucleic acid from the Cannabis plant with at least twocannabidiolic acid (CBDA) synthase primers under conditions sufficientfor amplification of a Bd variant of CBDA synthase (CBDA^(Bd)) genesequence. In some embodiments, at least one CBDA^(Bd) synthase primer iscomplementary to a sequence that bridges a 4 nucleotide deletion foundin the CBDA^(Bd) open reading frame.

In some embodiments, a method includes contacting a sample thatcomprises nucleic acid from the Cannabis plant with at least twocannabidiolic acid (CBDA) synthase primers under conditions sufficientfor amplification of a wild-type CBDA (CBDA^(WT)) synthase genesequence. In some embodiments, at least one CBDA^(WT) synthase primer iscomplementary to a sequence that includes the 4 nucleotide deleted inthe CBDA^(Bd) synthase open reading frame.

In some embodiments, a method includes a combination of steps thatinclude contacting a sample that comprises nucleic acid from theCannabis plant with primers under conditions sufficient foramplification of two or more of a THCA synthase gene sequence, a variantTHCA synthase gene sequence, a CBDA^(WT), synthase gene sequence, and/ora CBDA^(mut) synthase gene sequence.

In some embodiments, a method includes amplification of THCA synthasegene sequence that is at least 2000 nucleotide long. In some certainembodiments, an amplified THCA synthase gene sequences is 1500-2500,1750-2250, 2000-2200, or 2100-2200 nucleotides long. In someembodiments, an amplified THCA synthase gene sequence includes at leastpart of the THCA synthase promoter and 5′ UTR.

In some embodiments, amplification of one or more nucleic acid sequencesin a Cannabis genome is by isothermic nucleic acid amplification. Insome embodiments, amplification includes performing a Loop-MediatedIsothermal Amplification (LAMP) assay. In some embodiments, a LAMP assayis a colorimetric assay.

In some embodiments, a Cannabis plant is a C. sativa plant. In someembodiments, a Cannabis plant is a C. indica plant. In some embodiments,a Cannabis plant is a C. ruderalis plant.

In some embodiments, amplification of a THCA synthase gene sequenceincludes contacting a sample that comprises nucleic acid from theCannabis plant with at least five THCA synthase primers under conditionssufficient for LAMP of a THCA synthase gene sequence. In some certainembodiments, the at least five primer sequences are at least 70%, 75%,80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to each of SEQ ID NOs: 8-12.

In some embodiments, amplification of a mutated THCA synthase genesequence includes contacting a sample that comprises nucleic acid fromthe Cannabis plant with at least five THCA synthase primers underconditions sufficient for LAMP of a mutated THCA synthase gene sequence.

In some embodiments, amplification of a wild-type cannabidiolic acid(CBDA^(WT)) synthase gene sequence includes contacting a sample thatcomprises nucleic acid from the Cannabis plant with at least five CBDAsynthase primers under conditions sufficient for LAMP of a wild-typeCBDA (CBDA^(WT)) synthase gene sequence. In some certain embodiments,detecting the presence of a CBDA^(WT) synthase gene sequence is by LAMPamplification and includes contacting nucleic acid from a Cannabis plantwith six primers that are at least 70%, 75%, 80%, 85%, 85%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto each of SEQ ID NOs: 13-18 and 27.

In some embodiments, amplification of a mutated cannabidiolic acid(CBDA^(mut)) synthase gene sequence includes contacting a sample thatcomprises nucleic acid from the Cannabis plant with at least five CBDAsynthase primers under conditions sufficient for LAMP of a CBDA^(mut)synthase gene sequence. In some embodiments a CBDA^(mut) synthase is adeletion (CBDA^(del)). In some certain embodiments, detecting thepresence of a CBDA^(del) synthase gene sequence is by LAMP amplificationand includes contacting nucleic acid from a Cannabis plant with sixprimers that are at least 70%, 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to eachof SEQ ID NOs: 13, 15-19, and 27.

In some aspects, the present disclosure provides kits that includeprimers that are complementary to sequences encoding Cannabis enzymesthat are involved in cannabinoid synthesis. In some embodiments, a kitmay be used to amplify a genomic sequence form a C. sativa, C. indica,and/or a C. ruderalis plant.

In some embodiments, a kit includes at least two THCA synthase primersincluding a forward THCA synthase primer and a reverse THCA synthaseprimer, wherein the forward THCA synthase primer is complementary to asequence that is 200-1000 nucleotides upstream of a THCA synthase openreading frame in a Cannabis genome and the reverse THCA synthase primeris complementary to a sequence that is 50-1000 nucleotides downstream ofthe THCA synthase open reading frame.

In some embodiments, a forward THCA synthase primer is or comprises asequence complementary to 20-60 nucleotides of SEQ ID NO: 6 and whereinthe reverse THCA synthase primer is or comprises a sequencecomplementary to 20-60 nucleotides of SEQ ID NO: 7. In some embodiments,a kit includes at least five THCA synthase primers.

In some embodiments, a kit of the present disclosure may additionally oralternatively include at least two cannabidiolic acid (CBDA) synthaseprimers for amplification of a Bd variant of CBDA synthase (CBDA^(Bd))gene sequence and/or at least two cannabidiolic acid (CBDA) synthaseprimers for amplification of a wild-type CBDA (CBDA^(WT)) synthase genesequence. In some embodiments, at least one CBDA^(Bd) synthase primer iscomplementary to a sequence that bridges a 4 nucleotide deletion foundin the CBDA^(Bd) open reading frame. In some embodiments, at least oneCBDA^(WT) synthase primer is complementary to a sequence that includesthe 4 nucleotides deleted in the CBDA^(Bd) synthase open reading frame.

In some certain embodiments, at least one CBDA^(WT) synthase primer isor comprises a sequence that is at least 80% identical to SEQ ID NO: 14.In some certain embodiments, at least one CBDA^(Bd) synthase primer isor comprises a sequence that is at least 80% identical to SEQ ID NO: 19.In some certain embodiments, at least one CBDA^(WT) synthase primer isor comprises a sequence of SEQ ID NO: 14. In some certain embodiments,at least one CBDA^(Bd) synthase primer is or comprises a sequence of SEQID NO: 19.

In some embodiments, a kit of the present disclosure may additionally oralternatively include primers for amplification of one or more of awild-type olivetol synthase gene sequence, a variant olivetol synthasegene sequence, a wild-type divarinic acid synthase gene sequence, avariant divarinic acid synthase gene sequence, a wild-type limonenesynthase gene sequence, and a variant limonene synthase gene sequencefrom a sample that comprises Cannabis nucleic acid.

Any of the kits of the present disclosure may additionally includereagents for a Loop-Mediated Isothermal Amplication (LAMP) assay. Insome embodiments, a LAMP assay is a colorimetric LAMP assay.

These, and other aspects encompassed by the present disclosure, aredescribed in more detail below and in the claims.

BRIEF DESCRIPTION OF THE DRAWING

The Figures described below, that together make up the Drawing, are forillustration purposes only, not for limitation.

FIG. 1: Panels (A) and (B) provide schematic representations of portionof the cannabinoid synthesis pathway.

FIG. 2: depicts a subtype classification system for Cannabis plants.

FIG. 3: depicts an alignment of various primers with the sequence of aCannabis THCA synthase gene sequence (SEQ ID NO: 20). Exemplarymutations in a THCA synthase gene sequence are provided alongTHCAS-FIBER dotted underneath the ORF/CDS sequence.

FIG. 4: depicts a position-specific weight matrix for alignment of CBDAsynthase alleles (SEQ ID NOS: 23, 24, and 25) in a Cannabis plant.

FIG. 5: depicts isothermic (e.g., LAMP) amplification of a THCA synthasegene sequence. Panel (A) depicts an isothermic (e.g., LAMP)amplification at 65° C. of THCA synthase gene sequence with Kitamuraprimers (“K primers”) (Kitamura et al., (2017) Journal of naturalmedicines, 71(1):86-95 and Kitamura et al., (2016) Biological &pharmaceutical bulletin, 39(7):1144-9, both of which are incorporatedherein by reference) and Long Range primers (“LR primers”) on theCannabis strain Grandaddy Purple (“GDP”). Additionally, an isothermicamplification with K primers of various strains of Cannabis: Otto, twohemp samples, Grape Stomper (RSP10516), and Erk Train X Deadhead OG(RSP10517) were also determined. Panel (B) depicts an isothermic (LAMP)amplification with LR primers at various temperatures for multiplestrains of Cannabis.

FIG. 6: depicts an isothermic (LAMP) amplification of THCA gene sequenceat 65° C. with Long Range primers on various Cannabis strains.

FIG. 7: depicts isothermic (LAMP) amplification of (Panel (A)) a CBDAsynthase gene sequence and (Panel (B)) isothermic (LAMP) amplificationof THCA gene sequence on various hemp varietals of Cannabis. Panel (C)includes a map of the samples run in both Panels (A) and (B).

FIG. 8: depicts isothermic (LAMP) amplification assays for CBDA^(wt)(Panel (A)) and CBDA^(del) (Panel (B)) sequences on various hempvarietals of Cannabis.

FIG. 9: depicts exemplary primer sequences (SEQ ID NOS: 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 13, 19, 15, 16, 17, and 18 in that order)useful in the context of the present disclosure.

FIG. 10: depicts a table of exemplary THCA synthase gene sequence.

CERTAIN DEFINITIONS

Associated With: The term “associated with” is used herein to describean observed correlation between two items or events. For example, amutation in THCA synthase may be considered to be “associated with” aparticular cannabinoid synthesis profile and/or cannabinoid composition.

Cannabis: As used herein, “Cannabis” refers to any plant in the genusCannabis. In some embodiments, “Cannabis” refers to a part of, aspecific compound from, and/or any product from a Cannabis plant (e.g.,C. sativa, C. indica, C. ruderalis). For example, a “Cannabis enzyme”refers to an enzyme from a Cannabis plant. Similarly, a “Cannabisgenome” refers to a genome from a Cannabis plant. Cannabis includes both“marijuana” and “hemp,” two forms of Cannabis that are distinguished onthe basis of the relative abundances of different cannabinoids. Cannabisincludes any variety of Cannabis species, cultivar of Cannabis species,or hybrid between any Cannabis species.

Chemotype: As used herein, the term “chemotype” refers to chemicallydistinct entity (e.g., plant) with a particular profile of metabolites.In some embodiments, a chemotype is a particular type of Cannabis plantwith a particular profile of one or more cannabinoids. In someembodiments, plants having different chemotypes may have same ordifferent morphological characteristics. In some embodiments, achemotype is characterized by a highly abundant chemical produced bythat entity (e.g., plant). In some certain embodiments, a chemotype mayrefer to a Cannabis plant with an abundance of one or more cannabinoids(e.g., THC or CBD). In some embodiments, abundance of one or morecannabinoids may be a relative amount (e.g., a ratio of cannabinoids,such as a THC:CBD ratio).

Coding sequence: As used herein, the term “coding sequence” refers to asequence of a nucleic acid or its complement, or a part thereof, that(i) can be transcribed to an mRNA sequence that can be translated toproduce a polypeptide or a fragment thereof, or (ii) an mRNA sequencethat can be translated to produce a polypeptide or a fragment thereof.Coding sequences include exons in genomic DNA or immature primary RNAtranscripts, which are joined together by the cell's biochemicalmachinery to provide a mature mRNA.

Gene and Gene sequence: The term “gene,” as used herein, refers to apart of the genome that codes for a product (e.g., an RNA product and/ora polypeptide product). A “gene sequence” is a sequence that includes atleast a portion of a gene (e.g., all or part of a gene) and/orregulatory elements associated with a gene. In some embodiments, a geneincludes coding sequence; in some embodiments, a gene includesnon-coding sequence. In some particular embodiments, a gene may includeboth coding (e.g., exonic) and non-coding (e.g., intronic) sequences. Insome embodiments, a gene may include one or more regulatory elements(e.g., a promoter) that, for example, may control or impact one or moreaspects of gene expression (e.g., cell-type-specific expression,inducible expression, etc.).

Mutation: As used herein, the term “mutation” refers to a changeintroduced into a parental sequence, including, but not limited to,substitutions, insertions, deletions (including truncations). Theconsequences of a mutation include, but are not limited to, the creationof a new character, property, function, phenotype or trait not found inthe protein encoded by the parental sequence, or the increase orreduction/elimination of an existing character, property, function,phenotype or trait not found in the protein encoded by the parentalsequence.

Nucleic Acid: As used herein, the terms “nucleic acid,” “nucleic acidmolecule,” “oligonucleotide,” and “polynucleotide” are each used hereinto refer to a polymer of at least three nucleotides. In someembodiments, a nucleic acid comprises deoxyribonucleic acid (DNA). Insome embodiments comprises ribonucleic acid (RNA). In some embodiments,a nucleic acid is single stranded. In some embodiments, a nucleic acidis double stranded. In some embodiments, a nucleic acid comprises bothsingle and double stranded portions. Unless otherwise stated, the termsencompass nucleic acid-like structures with synthetic backbones, as wellas amplification products. In some embodiments, nucleic acids of thepresent disclosure are linear nucleic acids.

Plant part: As used herein, the term “plant part” refers to any part ofa plant including but not limited to the embryo, shoot, root, stem,seed, stipule, leaf, petal, flower bud, flower, ovule, bract, trichome,branch, petiole, internode, bark, pubescence, tiller, rhizome, frond,blade, ovule, pollen, stamen, and the like. The two main parts of plantsgrown in some sort of media, such as soil or vermiculite, are oftenreferred to as the “above-ground” part, also often referred to as the“shoots”, and the “below-ground” part, also often referred to as the“roots.” Plant part may also include certain extracts such as kief orhash which includes cannabis trichomes or glands.

Primer: The terms “primer,” as used herein, typically refers tooligonucleotides that hybridize in a sequence specific manner to acomplementary nucleic acid molecule (e.g., a nucleic acid moleculecomprising a target sequence). In some embodiments, a primer willcomprise a region of nucleotide sequence that hybridizes to at least 8,e.g., at least 10, at least 15, at least 20, at least 25, or 20 to 60nucleotides of a target nucleic acid (i.e., will hybridize to a sequenceof the target nucleic acid). In general, a primer sequence is identifiedas being either “complementary” (i.e., complementary to the coding orsense strand (+)), or “reverse complementary” (i.e., complementary tothe anti-sense strand (−)). In some embodiments, the term “primer” mayrefer to an oligonucleotide that acts as a point of initiation of atemplate-directed synthesis using methods such as PCR (polymerase chainreaction) under appropriate conditions (e.g., in the presence of fourdifferent nucleotide triphosphates and a polymerization agent, such asDNA polymerase in an appropriate buffer solution containing anynecessary reagents and at suitable temperature(s)). Such a templatedirected synthesis is also called “primer extension.” For example, aprimer pair may be designed to amplify a region of DNA using PCR. Such apair will include a “forward primer” and a “reverse primer” thathybridize to complementary strands of a DNA molecule and that delimit aregion to be synthesized and/or amplified.

Reference: As will be understood from context, a reference sequence,sample, population, agent or individual is one that is sufficientlysimilar to a particular sequence, sample, population, agent orindividual of interest to permit a relevant comparison (i.e., to becomparable). In some embodiments, information about a reference sampleis obtained simultaneously with information about a particular sample.In some embodiments, information about a reference sample is historical.In some embodiments, information about a reference sample is stored forexample in a computer-readable medium. In some embodiments, comparisonof a particular sample of interest with a reference sample establishesidentity with, similarity to, or difference of a particular sample ofinterest relative to a reference.

Regulatory Sequence: The term “regulatory sequence” is intended toinclude promoters, enhancers and other expression control elements(e.g., polyadenylation signals).

Wild type: As used herein, the term “wild-type” refers to a typical orcommon form existing in nature; in some embodiments it is the mostcommon form.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The disclosure provides useful methods for characterizing certaingenetic sequences of Cannabis plants. The present disclosure encompassesa recognition that amplification of a region of THCA synthase genesequence that includes a THCA synthase gene sequence promoter and/ortermination region can robustly predict the presence of a functionalTHCA synthase gene sequence. The present disclosure also provides amethod that reliably and accurately detects a CBDA synthase genesequence in a Cannabis plant. Methods of the present disclosure mayinclude characterization (e.g., detection, amplification) of CDBA genesequences, including wildtype (CDBA^(wt)) and deletion (CDBA^(del))sequences.

Cannabis Plants and Cannabinoids

Two sub-species of Cannabis plants are C. indica and C. sativa, whichare commonly distinguished based on morphology of a Cannabis plant.Generally, C. sativa plants are taller, loosely branched and have long,narrow leaves, while C. indica plants are shorter, more densely branchedand have wider leaves. However, there is some doubt about the accuracyof these generalizations. Originally, classification of C. indica wasmade in 1785 by a French biologist named Jean-Baptiste Lamarck whoobserved that certain marijuana plants from India were intoxicating andcould be made into hashish. In contrast, traditional hemp crops inEurope had little or no mind-altering effect. Lamark came up with thename C. indica to distinguish Indian cannabis from European hemp, whichwas known at the time as C. sativa. Therefore, additionally oralternatively, Cannabis plants can be characterized by production of oneor more chemical metabolites (e.g., cannabinoids). In some embodiments,a Cannabis plant is characterized as having a specified level (e.g.,high/low) of one or more cannabinoids, flavonoids and/or terpenes.

Cannabinoids are terpenophenolic secondary metabolites, produced byCannabis plants in the sessile and stalked trichomes. Trichomes aregenerally abundant on the inflorescences of a Cannabis plant, present inlower number on leaves, petioles and stems, and generally absent onroots and seeds. As a consequence, roots and seeds generally do notcontain cannabinoids. In some embodiments, a Cannabis plant for use in amethod of the present disclosure may include any plant part, such as,for example, a bloom, leaf, petiole, stem, root and/or seed.

As an annual, cannabis plants follow a solar cycle consisting of twobasic stages often referred to as vegetative, and bloom (flowering).Cannabinoid synthesis occurs predominantly in bloom (flowering) phase.In some embodiments, a Cannabis plant for use in a method of the presentdisclosure is in a vegetative state. In some embodiments, a Cannabisplant for use in a method of the present disclosure is in a floweringstate.

Cannabinoids such as, for example, delta-9-tetrahydrocannabinol (Δ9-THCor THC), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), andcannabichromene (CBC) have been identified from Cannabis plants. In someembodiments, a cannabinoid is an aryl-substituted monoterpene.Generally, cannabinoids are lipid soluble and neutral. Cannabinoids canbe divided into at least ten classes: cannabigerol, cannabichromene,cannabidiol, delta-9-tetrahydrocannabinol, delta-8-tetrahydrocannabinol,cannabicyclol, cannabielsoin, cannabinol and cannabinodiol, cannabitrioland miscellaneous cannabinoids.

In some embodiments, C. indica plants are characterized as having highTHC:CBD ratios and C. sativa plants are characterized as having highCBD:THC ratios. However, many strains produce varying amounts ofcannabinoids, which may be due to hybridization, or cross breeding.Accordingly, in some embodiments, a C. sativa plant may be rich in THCand a C. indica plant may have low THC.

Cannabis plants for use in accordance with the methods of the presentdisclosure may be, for example, a C. sativa plant and/or a C. indicaplant. In some embodiments, a Cannabis plant may be characterized asand/or determined to be rich in one or more cannabinoids such as acannabigerol, cannabichromene, cannabidiol,delta-9-tetrahydrocannabinol, delta-8-tetrahydrocannabinol,cannabicyclol, cannabielsoin, cannabinol and cannabinodiol, and/orcannabitriol. In some embodiments, a Cannabis plant is determined to beor characterized as rich in THC. In some embodiments, a Cannabis plantis determined to be or characterized as rich in CBD.

In some embodiments, a Cannabis plant may be characterized as and/ordetermined to be expressing a low amount of one or more cannabinoidssuch as a cannabigerol, cannabichromene, cannabidiol,delta-9-tetrahydrocannabinol, delta-8-tetrahydrocannabinol,cannabicyclol, cannabielsoin, cannabinol and cannabinodiol, and/orcannabitriol. In some embodiments, a Cannabis plant is determined to beor characterized as low in THC. In some embodiments, a Cannabis plant isdetermined to be or characterized as low in CBD.

The present disclosure also encompasses the recognition that levels oftwo or more cannabinoids may be related. FIG. 1 depict portions of thecannabinoid synthesis pathway. As shown in FIG. 1, Panels A and B,cannabigerolic acid is a precursor of both THCA and CBDA. THCA and CBDAeach undergo non-enzymatic conversion to THC and CBD, respectively.Since both THC and CBD are synthesized from the same precursor,increased synthesis of one of these compounds may reduce synthesis ofthe other (e.g., by depletion of precursor). In some embodiments, therate/amount of THC synthesis is inversely proportional to therate/amount of CBD synthesis. Moreover, as synthesis of both THC and CBDuse cannabigerolic acid, increased synthesis of either or both of thesecannabinoids may reduce the amount of cannabigerolic acid. In someembodiments, the rate/amount of THC synthesis is inversely proportionalto the rate/amount of cannabigerolic acid. In some embodiments, therate/amount of CBD synthesis is inversely proportional to therate/amount of cannabigerolic acid.

Earlier studies proposed a model that the genes coding for functionalTHCA- and CBDA-synthase (referred to as Bt and Bd, respectively) areallelic and codominant. (de Meijer et al. (2003) Genetics,163(1):335-46, PMID: 12586720, which is incorporated herein byreference). Variations in CBDA/THCA ratios could be directly related toa differential efficiency in transforming cannabigerolic acid by THCA-and CBDA-synthases. Id.

In some embodiments, a Cannabis plant genome includes a THCA synthasegene sequence. In some embodiments, a Cannabis plant genome includes awild-type THCA synthase gene sequence. In some embodiments, a Cannabisplant genome is homozygous for a wild-type THCA synthase gene sequence.In some embodiments, a Cannabis plant genome is heterozygous for awild-type THCA synthase gene sequence. In some embodiments, a Cannabisplant genome is homozygous for a variant THCA synthase gene sequence. Insome embodiments, a Cannabis plant genome is heterozygous for a variantTHCA synthase gene sequence. In some embodiments, a Cannabis plantgenome includes a THCA synthase gene sequence that is or comprises asequence that is 70%, 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, ora portion thereof.

Exemplary C. sativa THC Asynthase gene sequence SEQ ID NO: 1ATGGGAACCATAATAAACTATAAAAGTCATTATGTGTACTTGCTACCATAGGCACCTATATCCCACAAACTAGCTACCATAGCCAATTTCTTTTTTGTTTCCAATATCCAATTTTTATTGATGCCAAACTATTCAATGTACAATGTACATTTATTTTCAATAAGGGCTTCACCTAACAAAGGTGCCTAATTTTAGTTGATTTATTTTTTATCACATGTGACTATTTAATGACTATCAAATTATAAAATATTTAAGTCAATTTATTTGCCCCAACTCCAATATATAATATTATAAATAGGATAGTTCTCAATTCCTAATAATTCAAAAAATCATTAGGACTGAAGAAAAATGAATTGCTCAGCATTTTCCTTTTGGTTTGTTTGCAAAATAATATTTTTCTTTCTCTCATTCCATATCCAAATTTCAATAGCTAATCCTCGAGAAAACTTCCTTAAATGCTTCTCAAAACATATTCCCAACAATGTAGCAAATCCAAAACTCGTATACACTCAACACGACCAATTGTATATGTCTATCCTGAATTCGACAATACAAAATCTTAGATTCATCTCTGATACAACCCCAAAACCACTCGTTATTGTCACTCCTTCAAATAACTCCCATATCCAAGCAACTATTTTATGCTCTAAGAAAGTTGGCTTGCAGATTCGAACTCGAAGCGGTGGCCATGATGCTGAGGGTATGTCCTACATATCTCAAGTCCCATTTGTTGTAGTAGACTTGAGAAACATGCATTCGATCAAAATAGATGTTCATAGCCAAACTGCGTGGGTTGAAGCCGGAGCTACCCTTGGAGAAGTTTATTATTGGATCAATGAGAAGAATGAGAATCTTAGTTTTCCTGGTGGGTATTGCCCTACTGTTGGCGTAGGTGGACACTTTAGTGGAGGAGGCTATGGAGCATTGATGCGAAATTATGGCCTTGCGGCTGATAATATTATTGATGCACACTTAGTCAATGTTGATGGAAAAGTTCTAGATCGAAAATCCATGGGAGAAGATCTGTTTTGGGCTATACGTGGTGGTGGAGGAGAAAACTTTGGAATCATTGCAGCATGGAAAATCAAACTGGTTGATGTCCCATCAAAGTCTACTATATTCAGTGTTAAAAAGAACATGGAGATACATGGGCTTGTCAAGTTATTTAACAAATGGCAAAATATTGCTTACAAGTATGACAAAGATTTAGTACTCATGACTCACTTCATAACAAAGAATATTACAGATAATCATGGGAAGAATAAGACTACAGTACATGGTTACTTCTCTTCAATTTTTCATGGTGGAGTGGATAGTCTAGTCGACTTGATGAACAAGAGCTTTCCTGAGTTGGGTATTAAAAAAACTGATTGCAAAGAATTTAGCTGGATTGATACAACCATCTTCTACAGTGGTGTTGTAAATTTTAACACTGCTAATTTTAAAAAGGAAATTTTGCTTGATAGATCAGCTGGGAAGAAGACGGCTTTCTCAATTAAGTTAGACTATGTTAAGAAACCAATTCCAGAAACTGCAATGGTCAAAATTTTGGAAAAATTATATGAAGAAGATGTAGGAGCTGGGGTGTTGTACCCTTACGGTGGTATAATGGAGGAGATTTCAGAATCAGCAATTCCATTCCCTCATCGAGCTGGAATAATGTATGAACTTTGGTACACTGCTTCCTGGGAGAAGCAAGAAGATAATGAAAAGCATATAAACTGGGTTCGAAGTGTTTATAATTTTACGACTCCTTATGTGTCCCAAAATCCAAGATTGGCGTATCTCAATTATAGGGACCTTGATTTAGGAAAAACTAATCATGCGAGTCCTAATAATTACACACAAGCACGTATTTGGGGTGAAAAGTATTTTGGTAAAAATTTTAACAGGTTAGTTAAGGTGAAAACTAAAGTTGATCCCAATAATTTTTTTAGAAACGAACAAAGTATCCCACCTCTTCCACCGCATCATCATTAATTATCTTTAAATAGATATATTTCCCTTATCAATTAGTTAATCATTATACCATACATACATTTATTGTATATAGTTTATCTACTCATATTATGTATGCTCCCAAGTATGAAAATCTACATT AGAACTGTGTAGACAATCATA

In some embodiments, a Cannabis plant genome includes a CBDA synthasegene sequence. In some embodiments, a Cannabis plant genome includes awild-type CBDA synthase gene sequence. In some embodiments, a Cannabisplant genome is homozygous for a wild-type CBDA synthase gene sequence.In some embodiments, a Cannabis plant genome is heterozygous for awild-type CBDA synthase gene sequence. In some embodiments, a Cannabisplant genome is homozygous for a variant CBDA synthase gene sequence. Insome embodiments, a Cannabis plant genome is heterozygous for a variantCBDA synthase gene sequence. In some embodiments, a variant CBDAsynthase gene sequence comprises a deletion. In some embodiments, avariant CBDA synthase gene sequence comprises a deletion of a sequencecomprising CGTA (SEQ ID NO:3). In some embodiments, a Cannabis plantgenome includes a CBDA synthase gene sequence that is or comprises asequence that is 70%, 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, ora portion thereof.

Exemplary C. sativa CBDA synthase gene sequence SEQ ID NO: 2GATATATATCTCACACGGATGCACCTAACAATGATGCCTAATTTTTGTGAATTTTTTTTACCACATGACTTAATGATATCAAATTATGAAATATTTAGTTAATTTATTTGCCCCTGCTCCAATATATAAAGCTATAAATAGGATAGTTCTTAATCCATAGTAATTCAAAATTCATTAGAACTAAAGAAAAATGAAGTGCTCAACATTCTCCTTTTGGTTTGTTTGCAAGATAATATTTTTCTTTTTCTCATTCAATATCCAAACTTCCATTGCTAATCCTCGAGAAAACTTCCTTAAATGCTTCTCGCAATATATTCCCAATAATGCAACAAATCTAAAACTCGTATACACTCAAAACAACCCATTGTATATGTCTGTCCTAAATTCGACAATACACAATCTTAGATTCACCTCTGACACAACCCCAAAACCACTTGTTATCGTCACTCCTTCACATGTCTCTCATATCCAAGGCACTATTCTATGCTCCAAGAAAGTTGGCTTGCAGATTCGAACTCGAAGTGGTGGTCATGATTCTGAGGGCATGTCCTACATATCTCAAGTCCCATTTGTTATAGTAGACTTGAGAAACATGCGTTCAATCAAAATAGATGTTCATAGCCAAACTGCATGGGTTGAAGCCGGAGCTACCCTTGGAGAAGTTTATTATTGGGTTAATGAGAAAAATGAGAATCTTAGTTTGGCGGCTGGGTATTGCCCTACTGTTTGCGCAGGTGGACACTTTGGTGGAGGAGGCTATGGACCATTGATGAGAAACTATGGCCTCGCGGCTGATAATATCATTGATGCACACTTAGTCAACGTTCATGGAAAAGTGCTAGATCGAAAATCTATGGGGGAAGATCTCTTTTGGGCTTTACGTGGTGGTGGAGCAGAAAGCTTCGGAATCATTGTAGCATGGAAAATTAGACTGGTTGCTGTCCCAAAGTCTACTATGTTTAGTGTTAAAAAGATCATGGAGATACATGAGCTTGTCAAGTTAGTTAACAAATGGCAAAATATTGCTTACAAGTATGACAAAGATTTATTACTCATGACTCACTTCATAACTAGGAACATTACAGATAATCAAGGGAAGAATAAGACAGCAATACACACTTACTTCTCTTCAGTTTTCCTTGGTGGAGTGGATAGTCTAGTCGACTTGATGAACAAGAGTTTTCCTGAGTTGGGTATTAAAAAAACGGATTGCAGACAATTGAGCTGGATTGATACTATCATCTTCTATAGTGGTGTTGTAAATTACGACACTGATAATTTTAACAAGGAAATTTTGCTTGATAGATCCGCTGGGCAGAACGGTGCTTTCAAGATTAAGTTAGACTACGTTAAGAAACCAATTCCAGAATCTGTATTTGTCCAAATTTTGGAAAAATTATATGAAGAAGATATAGGAGCTGGGATGTATGCGTTGTACCCTTACGGTGGTATAATGGATGAGATTTCAGAATCAGCAATTCCATTCCCTCATCGAGCTGGAATCTTGTATGAGTTATGGTACATATGTAGTTGGGAGAAGCAAGAAGATAACGAAAAGCATCTAAACTGGATTAGAAATATTTATAACTTCATGACTCCTTATGTGTCCAAAAATCCAAGATTGGCATATCTCAATTATAGAGACCTTGATATAGGAATAAATGATCCCAAGAATCCAAATAATTACACACAAGCACGTATTTGGGGTGAGAAGTATTTTGGTAAAAATTTTGACAGGCTAGTAAAAGTGAAAACCCTGGTTGATCCCAATAACTTTTTTAGAAACGAACAAAGCATCCCACCTCTTCCACGGCATCGTCATTAATGATCTTAAATAGATCTTTTTCTCTTATTAATTAGTCCTTATAATATACATATATTGATTATATATATAAAAATAGTTTGTCCGGGTGTACTGTGTATGCGATATATATCTCACAC

In some embodiments, a Cannabis plant genome includes an olivetolsynthase gene sequence. In some embodiments, a Cannabis plant genomeincludes a wild-type olivetol synthase gene sequence. In someembodiments, a Cannabis plant genome is homozygous for a wild-typeolivetol synthase gene sequence. In some embodiments, a Cannabis plantgenome is heterozygous for a wild-type olivetol synthase gene sequence.In some embodiments, a Cannabis plant genome is homozygous for a variantolivetol synthase gene sequence. In some embodiments, a Cannabis plantgenome is heterozygous for a variant olivetol synthase gene sequence. Insome embodiments, a Cannabis plant genome includes an olivetol synthasegene sequence that is or comprises a sequence that is 70%, 75%, 80%,85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 4, or a portion thereof.

Exemplary C. sativa olivetol synthase gene sequence SEQ ID NO: 4ATGAATCATCTTCGTGCTGAGGGTCCGGCCTCCGTTCTCGCCATTGGCACCGCCAATCCGGAGAACATTTTATTACAAGATGAGTTTCCTGACTACTATTTTCGCGTCACCAAAAGTGAACACATGACTCAACTCAAAGAAAAGTTTCGAAAAATATGTGACAAAAGTATGATAAGGAAACGTAACTGTTTCTTAAATGAAGAACACCTAAAGCAAAACCCAAGATTGGTGGAGCACGAGATGCAAACTCTGGATGCACGTCAAGACATGTTGGTAGTTGAGGTTCCAAAACTTGGGAAGGATGCTTGTGCAAAGGCCATCAAAGAATGGGGTCAACCCAAGTCTAAAATCACTCATTTAATCTTCACTAGCGCATCAACCACTGACATGCCCGGTGCAGACTACCATTGCGCTAAGCTTCTCGGACTGAGTCCCTCAGTGAAGCGTGTGATGATGTATCAACTAGGCTGTTATGGTGGTGGAACCGTTCTACGCATTGCCAAGGACATAGCAGAGAATAACAAAGGCGCACGAGTTCTCGCCGTGTGTTGTGACATAATGGCTTGCTTGTTTCGTGGGCCTTCAGAGTCTGACCTCGAATTACTAGTGGGACAAGCTATCTTTGGTGATGGGGCTGCTGCGGTGATTGTTGGAGCTGAACCCGATGAGTCAGTTGGGGAAAGGCCGATATTTGAGTTGGTGTCAACTGGGCAAACAATCTTACCAAACTCGGAAGGAACTATTGGGGGACATATAAGGGAAGCAGGACTGATATTTGATTTACATAAGGATGTGCCTATGTTGATCTCTAATAATATTGAGAAATGTTTGATTGAGGCATTTACTCCTATTGGGATTAGTGATTGGAACTCCATATTTTGGATTACACACCCAGGTGGGAAAGCTATTTTGGACAAAGTGGAGGAGAAGTTGCATCTAAAGAGTGATAAGTTTGTGGATTCACGTCATGTGCTGAGTGAGCATGGGAATATGTCTAGCTCAACTGTCTTGTTTGTTATGGATGAGTTGAGGAAGAGGTCGTTGGAGGAAGGGAAGTCTACCACTGGAGATGGATTTGAGTGGGGTGTTCTTTTTGGGTTTGGACCAGGTTTGACTGTCGAAAGAGTGGTCGTGCGTAGTGTTCCCATCA AATATTAA

In some embodiments, a Cannabis plant genome includes a divarinic acidsynthase gene sequence. In some embodiments, a Cannabis plant genomeincludes a wild-type divarinic acid synthase gene sequence. In someembodiments, a Cannabis plant genome is homozygous for a wild-typedivarinic acid synthase gene sequence. In some embodiments, a Cannabisplant genome is heterozygous for a wild-type divarinic acid synthasegene sequence. In some embodiments, a Cannabis plant genome ishomozygous for a variant divarinic acid synthase gene sequence. In someembodiments, a Cannabis plant genome is heterozygous for a variantdivarinic acid synthase gene sequence.

In some embodiments, a Cannabis plant genome includes a limonenesynthase gene sequence. In some embodiments, a Cannabis plant genomeincludes a wild-type limonene synthase gene sequence. In someembodiments, a Cannabis plant genome is homozygous for a wild-typelimonene synthase gene sequence. In some embodiments, a Cannabis plantgenome is heterozygous for a wild-type limonene synthase gene sequence.In some embodiments, a Cannabis plant genome is homozygous for a variantlimonene synthase gene sequence. In some embodiments, a Cannabis plantgenome is heterozygous for a variant limonene synthase gene sequence. Insome embodiments, a Cannabis plant genome includes a limonene synthasegene sequence that is or comprises a sequence that is 70%, 75%, 80%,85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 5, or a portion thereof.

Exemplary C. sativa limonene synthase gene sequence SEQ ID NO: 5ATGCAGTGCATAGCTTTTCACCAATTTGCTTCATCATCATCCCTCCCTATTTGGAGTAGTATTGATAATCGTTTTACACCAAAAACTTCTATTACTTCTATTTCAAAACCAAAACCAAAACTAAAATCAAAATCAAACTTGAAATCGAGATCGAGATCAAGTACTTGCTACTCCATACAATGTACTGTGGTCGATAACCCTAGTTCTACGATTACTAATAATAGTGATCGAAGATCAGCCAACTATGGACCTCCCATTTGGTCTTTTGATTTTGTTCAATCTCTTCCAATCCAATATAAGGGTGAATCTTATACAAGTCGATTAAATAAGTTGGAGAAAGATGTGAAAAGGATGCTAATTGGAGTGGAAAACTCTTTAGCCCAACTTGAACTAATTGATACAATACAAAGACTTGGAATATCTTATCGTTTTGAAAATGAAATCATTTCTATTTTGAAAGAAAAATTCACCAATAATAATGACAACCCTAATCCTAATTATGATTTATATGCTACTGCTCTCCAATTTAGGCTTCTACGCCAATATGGATTTGAAGTACCTCAAGAAATTTTCAATAATTTTAAAAATCACAAGACAGGAGAGTTCAAGGCAAATATAAGTAATGATATTATGGGAGCATTGGGCTTATATGAAGCTTCATTCCATGGGAAAAAGGGTGAAAGTATTTTGGAAGAAGCAAGAATTTTCACAACAAAATGTCTCAAAAAATACAAATTAATGTCAAGTAGTAATAATAATAATATGACATTAATATCATTATTAGTGAATCATGCTTTGGAGATGCCACTTCAATGGAGAATCACAAGATCAGAAGCTAAATGGTTTATTGAAGAAATATATGAAAGAAAACAAGACATGAATCCAACTTTACTTGAGTTTGCCAAATTGGATTTCAATATGCTGCAATCAACATATCAAGAGGAGCTCAAAGTACTCTCTAGGTGGTGGAAGGATTCTAAACTTGGAGAGAAATTGCCTTTCGTTAGAGATAGATTGGTGGAGTGTTTCTTATGGCAAGTTGGAGTAAGATTTGAGCCACAATTCAGTTACTTTAGAATAATGGATACAAAACTCTATGTTCTATTAACAATAATTGATGATATGCATGACATTTATGGAACATTGGAGGAACTACAACTTTTCACTAATGCTCTTCAAAGATGGGATTTGAAAGAATTAGATAAATTACCAGATTATATGAAGACAGCTTTCTACTTTACATACAATTTCACAAATGAATTGGCATTTGATGTATTACAAGAACATGGTTTTGTTCACATTGAATACTTCAAGAAACTGATGGTAGAGTTGTGTAAACATCATTTGCAAGAGGCAAAATGGTTTTATAGTGGATACAAACCAACATTGCAAGAATATGTTGAGAATGGATGGTTGTCTGTGGGAGGACAAGTTATTCTTATGCATGCATATTTCGCTTTTACAAATCCTGTTACCAAAGAGGCATTGGAATGTCTAAAAGACGGTCATCCTAACATAGTTCGCCATGCATCGATAATATTACGACTTGCAGATGATCTAGGAACATTGTCGGATGAACTGAAAAGAGGCGATGTTCCTAAATCAATTCAATGTTATATGCACGATACTGGTGCTTCTGAAGATGAAGCTCGTGAGCACATAAAATATTTAATAAGTGAATCATGGAAGGAGATGAATAATGAAGATGGAAATATTAACTCTTTTTTCTCAAATGAATTTGTTCAAGTTTGCCAAAATCTTGGTAGAGCGTCACAATTCATATACCAGTATGGCGATGGACATGCTTCTCAGAATAATCTATCGAAAGAGCGCGTTTTAGGGTTGATTAT TACTCCTATCCCCATGTAA

Classification of Cannabis

Provided herein is a recognition that certain genotypes are associatedwith altered cannabinoid synthesis or an altered cannabinoid profile.Genetic information can positively benefit consumers, growers, andregulators of Cannabis. There are at least several hundred compoundsfound in Cannabis and currently only a fraction of these compounds areanalyzed. As used herein, chemotype of a Cannabis plant refers to anabundance and/or deficiency of one or more compounds found in a Cannabisplant (e.g., cannabinoids, flavonoids and/or terpenes). In someembodiments, a chemotype is an abundance of one or more cannabinoids,flavonoids and/or terpenes. In some embodiments, a chemotype is arelative amount of two or more cannabinoids, flavonoids and/or terpenes.

Chemotype determination has been limited as quantitative analyticaltechniques and standards do not currently exist for all Cannabiscompounds. As a result, previously described chemotypes fail to capturethe chemical and potential medicinal complexity in a Cannabis plant. Thepresent disclosure encompasses a recognition that genetic analysis, forexample, of one, two, three, four, five or more synthetic enzymes, canserve as a proxy for unmeasured chemical complexity.

The present disclosure encompasses the recognition that genetic analysiscan be used to assess the potential of a Cannabis plant to producecertain compounds, such as cannabinoids, flavonoids and/or terpenes.While a Cannabis plant can be raised under conditions that result invaried expression and/or concentrations of certain compounds, therelative ratios of key chemotypic gene sequences, such as, for example,THCA synthase and CBDA synthase, are usually genetically determined. Forexample, there are currently no known agricultural methods to make aCBD-dominant Cannabis strain become a THC-dominant Cannabis strain viaenvironmental conditions. These critical chemotypes are governed bygenetic alterations in their respective enzymatic synthases (e.g., CDBAsynthase and THCA synthase).

In some embodiments, methods of the present disclosure include geneticanalysis of one, two, three, four, five or more enzymes involved in theproduction of cannabinoids, flavonoids and/or terpenes. In someembodiments, methods of the present disclosure include amplification ofa genomic region that encodes an enzyme involved in the production of acannabinoid, flavonoid and/or terpene. In some embodiments, methods ofthe present disclosure include amplification of a plurality of genomicregions that encode enzymes involved in the production of a cannabinoid,flavonoid and/or terpene. In some embodiments, methods includeamplification of a portion of a gene sequence that encodes an enzymeinvolved in the production of a cannabinoid, flavonoid and/or terpene.

Genetic prediction of cannabinoid production has been an active area ofstudy. See, for example, Weiblen et al., (2015), The New Phytologist,PMID: 26189495; Marks et al., (2009) Journal of Experimental Botany,60(13):3715-26, PMID: 19581347; Onofri et al., (2015) Phytochemistry,116:57-68, PMID: 25865737; de Meijer et al. (2003) Genetics,163(1):335-46, PMID: 12586720; and Kojoma et al. (2006), Forensic Sci.Int, 159(2-3):132-40, PMID: 16143478, each of which is incorporatedherein by reference. However, there are significant caveats andshortcomings with prior methods of genetic prediction of cannabinoidproduction. For example, while alleles of Bt (functional THCA synthase)and Bd (functional CBDA synthase) have been proposed as a model forgenerating Type I, II, III, and IV plants, the exact DNA sequences thatgovern these alleles remain unknown. As described in the examplesherein, single molecule sequencing was performed to identify mutationsin gene sequences in cannabinoid synthesis pathway enzyme(s) (e.g., THCAsynthase and CBDA synthase). This sequencing revealed a more refinedstructure of inheritance and numerous additional subtypes that can beidentified by genotyping to predict chemical inheritance. See, FIG. 2.

Kitamura describes amplification of a small region of THCA synthase toresolve hemp from drug-type (i.e., THC expressing) strains. Kitamura etal., (2017) Journal of natural medicines, 71(1):86-95, PMID: 27535292and Kitamura et al., (2016) Biological & pharmaceutical bulletin,39(7):1144-9, PMID: 27118244, each of which is incorporated herein byreference. However, the present disclosure recognizes that these studiesonly tested 3 cultivars and also failed to consider CBDA status. Thepresent disclosure encompasses a recognition that an understanding CBDAstatus is critical for determining allelic balance of CBDA and THCAsynthases, which may be important for Cannabis breeding efforts.

Cannabis genomes are about 10 fold more variable than human genomes.Moreover, a given Cannabis strain is often capable of crossing withhighly divergent Cannabis strains (e.g., Cannabis strains of a differentchemotype, genotype, etc.). As a result, even sibling strains cannot benot be assumed to have the same chemotype. The present disclosureprovides methods and kits suitable for genetic characterization ofindividual Cannabis plants.

In some embodiments, a method includes detection of one or a pluralityof sequences that include a gene sequence or portion thereof in a samplethat comprises nucleic acid from a Cannabis plant. In some embodiments,a gene sequence encodes an enzyme involved in the synthesis of one ormore cannabinoids.

In some embodiments, a method of the present disclosure includesdetecting the presence of a tetrahydrocannabinolic acid (THCA) synthasegene sequence or a portion thereof. The present disclosure encompassesthe recognition that THCA synthase is under selective breeding pressureand has approximately a 4 fold higher polymorphism rate than a Cannabisgenome in general. Moreover, a THCA synthase gene sequence can include aSNP approximately every 25 nucleotides, which complicates design ofprimers that anneal within the open reading frame of THCA synthase. Insome embodiments, a THCA synthase gene sequence encodes a wildtype THCAsynthase enzyme (i.e., functional). In some embodiments, a THCA synthasegene sequence includes a THCA synthase gene sequence promoter sequence.In some embodiments, a THCA synthase gene sequence includes a THCAsynthase gene sequence 5′ UTR sequence.

In some embodiments, a method of the present disclosure includesamplifying a THCA synthase gene sequence or portion thereof from asample that comprises nucleic acid from a Cannabis plant. In someembodiments, amplification of a THCA synthase gene sequence includescontacting nucleic acid from a Cannabis plant with a forward THCAsynthase primer that is complementary to a sequence that is 200-1000nucleotides upstream of a THCA open reading frame. In some embodiments,a forward THCA synthase primer is complementary to a sequence that is200 to 800 nucleotides, 200 to 600 nucleotides, 200 to 400 nucleotidesupstream of a THCA open reading frame. In some certain embodiments, aforward THCA synthase primer is complementary to a sequence that is atleast 70%, 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of SEQ ID NO: 6.In some certain embodiments, a forward THCA synthase primer is at 20 to60 nucleotides long.

Exemplary C. sativa sequence upstream of THCAsynthase open reading frame SEQ ID NO: 6CATAGCGACTATCGTGATGGGAACCATAATAAACTATAAAAGTCATTATGTGTACTTGCTACCATAGGCACCTATATCCCACAAACTAGCTACCATAGCCAATTTCTTTTTTGTTTCCAATATCCAATTTTTATTGATGCCAAACTATTCAATGTACAATGTACATTTATTTTCAATAAGGGCTTCACCTAACAAAGGTGCCTAATTTTAGTTGATTTATTTTTTATCACATGTGACTATTTAATGACTATCAAATTATAAAATATTTAAGTCAATTTATTTGCCCCAACTCCAATATATAATATTATAAATAGGATAGTTCTCAATTCCTAATAATTCAAAA AATCATTA

In some embodiments, amplification of a THCA synthase gene sequenceincludes contacting nucleic acid from a Cannabis plant with a reverseTHCA synthase primer that is complementary to a sequence that is200-1000 nucleotides downstream of a THCA open reading frame. In someembodiments, a reverse THCA synthase primer is complementary to asequence that is 200 to 800 nucleotides, 200 to 600 nucleotides, 200 to400 nucleotides downstream of a THCA open reading frame. In some certainembodiments, a reverse THCA synthase primer is complementary to asequence that is at least 70%, 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to aportion of SEQ ID NO: 7. In some certain embodiments, a reverse THCAsynthase primer is about 20 to 60 nucleotides long.

Exemplary C. sativa sequence downstream of THCAsynthase open reading frame SEQ ID NO: 7TTATCAATTAGTTAATCATTATACCATACATACATTTATTGTATATAGTTTATCTACTCATATTATGTATGCTCCCAAGTATGAAAATCTACATTAGAACTGTGTAGACAATCATACATAGCGACTATCGTG

In some embodiments, amplification of a THCA synthase gene sequenceincludes contacting nucleic acid from a Cannabis plant with two or moreprimers that are at least 70%, 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any ofSEQ ID NOs: 8-12.

In some embodiments, a method of the present disclosure includesdetecting the presence of a variant THCA synthase gene sequence in asample that comprises nucleic acid from the Cannabis plant. In someembodiments, a method of the present disclosure includes amplifying avariant THCA synthase gene sequence or portion thereof from a samplethat comprises nucleic acid from a Cannabis plant.

In some embodiments, a method of the present disclosure includesdetecting the presence of a mutated THCA synthase gene sequence in asample that comprises nucleic acid from the Cannabis plant. In someembodiments, a mutated THCA synthase gene sequence includes at least onemutation that alters the activity of a THCA synthase encoded by themutated THCA synthase gene sequence relative to a THCA synthase encodedby a wild-type THCA synthase gene sequence.

In some embodiments, a method of the present disclosure includesdetecting the presence of a polymorphism within a THCA synthase genesequence in a sample that comprises nucleic acid from the Cannabisplant. In some embodiments, a polymorphism within a THCA synthase genesequence is or comprises one or more variants as described in FIG. 3and/or FIG. 10. FIG. 10 prevents a chart describing certain SingleNucleotide Polymorphism (SNP) variants of THCA synthase in differentCannabis plant strains (indicated by accession code). SNPs that resultin an amino acid change are indicated in bold, and the type of change iswith the corresponding position in the amino acid sequence is indicatedin the bottom row. In the last column is provided the proportion ofTHC(V)A product of the total cannabinoid fraction that is accumulated ineach Cannabis plant variant. In some embodiments, a THCA synthase genesequence is or comprises a I63L variant, a V125L variant, a E236Qvariant, a A250D variant, a E265G variant, and/or a G410Evariant. Insome certain embodiments, a THCA synthase gene sequence is or comprisesa A411V variant.

In some embodiments, a method of the present disclosure includesdetecting the presence of a wild-type cannabidiolic acid (CBDA^(WT))synthase gene sequence in a sample that comprises nucleic acid from theCannabis plant. In some embodiments, a method of the present disclosureincludes amplifying a CBDA^(WT) synthase gene sequence or portionthereof from a sample that comprises nucleic acid from a Cannabis plant.

In some embodiments, a method of the present disclosure includesdetecting the presence of a mutated cannabidiolic acid (CBDA^(mut))synthase gene sequence in a sample that comprises nucleic acid from theCannabis plant, wherein the mutated CBDA synthase gene sequencecomprises a deletion mutation. In some embodiments, a CBDA^(del)sequence includes or comprises a deletion of CGTA (SEQ ID NO:3). In someembodiments, a method of the present disclosure includes amplifying aCBDA^(mut) synthase gene sequence or portion thereof from a sample thatcomprises nucleic acid from a Cannabis plant. In some embodiments, amethod of the present disclosure includes amplifying a CBDA^(del)synthase gene sequence or portion thereof from a sample that comprisesnucleic acid from a Cannabis plant.

In some embodiments, a method of the present disclosure includesdetecting a combination of two or more genomic sequences selected from:a THCA synthase gene sequence, a variant or mutant THCA synthase genesequence, a CBDA^(WT) synthase, and CBDA^(mut) synthase gene sequence.In some embodiments, a method of the present disclosure includesamplifying two or more genomic sequences selected from: a THCA synthasegene sequence, a variant or mutant THCA synthase gene sequence, aCBDA^(WT) synthase, and CBDA^(mut) synthase gene sequence, from a samplethat comprises nucleic acid from a Cannabis plant.

In some embodiments, a method of the present disclosure includesdetecting the presence of an olivetol synthase gene sequence in a samplethat comprises nucleic acid from a Cannabis plant. In some embodiments,a method of the present disclosure includes amplifying an olivetolsynthase gene sequence or portion thereof from a sample that comprisesnucleic acid from a Cannabis plant. In some embodiments, a method of thepresent disclosure includes detecting the presence of a variant olivetolsynthase gene sequence in a sample that comprises nucleic acid from aCannabis plant. In some embodiments, a method of the present disclosureincludes amplifying a variant olivetol synthase gene sequence or portionthereof from a sample that comprises nucleic acid from a Cannabis plant.

In some embodiments, a method of the present disclosure includesdetecting the presence of a divarinic acid synthase gene sequence in asample that comprises nucleic acid from a Cannabis plant. In someembodiments, a method of the present disclosure includes amplifying adivarinic acid synthase gene sequence or portion thereof from a samplethat comprises nucleic acid from a Cannabis plant. In some embodiments,a method of the present disclosure includes detecting the presence of avariant divarinic acid synthase gene sequence in a sample that comprisesnucleic acid from a Cannabis plant. In some embodiments, a method of thepresent disclosure includes amplifying a variant divarinic acid synthasegene sequence or portion thereof from a sample that comprises nucleicacid from a Cannabis plant.

In some embodiments, a method of the present disclosure includesdetecting the presence of a limonene synthase gene sequence in a samplethat comprises nucleic acid from a Cannabis plant. In some embodiments,a method of the present disclosure includes amplifying a limonenesynthase gene sequence or portion thereof from a sample that comprisesnucleic acid from a Cannabis plant. In some embodiments, a method of thepresent disclosure includes detecting the presence of a variant limonenesynthase gene sequence in a sample that comprises nucleic acid from aCannabis plant. In some embodiments, a method of the present disclosureincludes amplifying a variant limonene synthase gene sequence or portionthereof from a sample that comprises nucleic acid from a Cannabis plant.

In some embodiments, a method of the present disclosure includesdetecting a combination of two, three, four, five, six, seven, eight, ormore genomic sequences selected from: a THCA synthase gene sequence, avariant or mutant THCA synthase gene sequence, a CBDA^(WT) synthase, andCBDA^(mut) synthase gene sequence. In some embodiments, a method of thepresent disclosure includes amplifying two or more genomic sequencesselected from: a THCA synthase gene sequence, a variant or mutant THCAsynthase gene sequence, a CBDA^(WT) synthase, a CBDA^(mut) synthase genesequence, a olivetol synthase gene sequence, a variant olivetol synthasegene sequence, a divarinic acid synthase gene sequence, a variantdivarinic acid synthase gene sequence, a limonene synthase genesequence, and a variant limonene synthase gene sequence from a samplethat comprises nucleic acid from the Cannabis plant.

Methods for Genotyping Cannabis

In vitro nucleic acid amplification technique can be used for genotypingCannabis plants. In vitro nucleic acid amplification techniques can begrouped according to the temperature requirements of the procedure. Forexample, polymerase chain reaction (PCR) is the most popular method as atechnique of amplifying nucleic acid in vitro. PCR has high sensitivitybased on the effect of exponential amplification. Further, since a PCRamplification product can be recovered as DNA, this method is appliedwidely for genetic engineering techniques such as gene sequence cloningand structural determination. In PCR, however, temperature cycling or aspecial temperature controller is necessary for practice; theexponential progress of the amplification reaction causes a problem inquantification. Other PCR-based amplification techniques include, forexample, transcription-based amplification (D. Y. Kwoh, et at. 1989.Proc. Natl. Acad Sci. USA 86, 1173-1177, which is incorporated herein byreference), ligase chain reaction (LCR; D. Y. Wu, et al. 1989. Genomics4, 560-569; K. Barringer, et al. 1990. Gene 89, 117-122; F. Barany.1991. Proc. Natl. Acad. Sci. USA 88, 189-193, each of which areincorporated herein by reference), and restriction amplification (U.S.Pat. No. 5,102,784, which is incorporated herein by reference).

More recently, a number of isothermal nucleic acid amplificationtechniques have been developed. That is, these techniques do not rely onthermocycling to drive nucleic acid amplification. Isothermalamplification techniques typically utilize DNA polymerases withstrand-displacement activity, thus eliminating the high temperature meltcycle that is required for PCR. This allows isothermal techniques to befaster and more energy efficient than PCR, and also allows for moresimple and thus lower cost instrumentation since rapid temperaturecycling is not required. For example, methods such as StrandDisplacement Amplification (SDA; Walker, et at., (1992) Proc. Natl.Acad. Sci. USA 89: 392-396; Walker, et al., (1992) Nuc. Acids. Res.20:1691-1696; U.S. Pat. No. 5,648,211 and EP 0 497 272, each of which isincorporated herein by reference); self-sustained sequence replication(3SR; J. C. Guatelli, et al., (1990) Proc. Natl. Acad. Sci. USA, 87:1874-1878, which is incorporated herein by reference); and Qβ replicasesystem (Lizardi, et al., (1988) BioTechnology, 6: 1197-1202, which isincorporated herein by reference) are isothermal reactions. See also,Nucleic Acid Isothermal Amplification Technologies—A Review.Nucleosides, Nucleotides and Nucleic Acids (2008) v27(3):224-243, whichis incorporated herein by reference.

In some embodiments, an in vitro nucleic acid amplification assay foruse in the methods of the present disclosure is an isothermalamplification method. Isothermal amplification methods include, forexample, transcription mediated amplification (TMA) or self-sustainedsequence replication (3SR), nucleic acid sequence-based amplification(NASBA), signal mediated amplification of RNA technology (SMART), stranddisplacement amplification (SDA), rolling circle amplification (RCA),loop-mediated isothermal amplification of DNA (LAMP), isothermalmultiple displacement amplification (IMDA), helicase-dependentamplification (HDA), single primer isothermal amplification (SPIA), andcircular helicase-dependent amplification (cHDA))

LAMP Assay

It is envisioned that LAMP amplification assays as described herein willprovides a rapid and scalable means to resolve chemotypes of Cannabisplants. In some embodiments, an in vitro nucleic acid amplificationassay for use in the methods of the present disclosure is Loop-MediatedIsothermal Amplification (LAMP). Typically, LAMP reactions use astrand-displacing DNA polymerase with four to six primers, which canresult in exponential amplification of a target sequence.

In LAMP, a target sequence is amplified at a constant temperature. Insome embodiments, a LAMP reaction temperature is between 37° C. and 75°C. In some embodiments, a LAMP reaction temperature is between 50° C.and 70° C. In some embodiments, a LAMP reaction temperature is between55° C. and 65° C. In some embodiments, a LAMP reaction temperature isbetween 60° C. and 65° C. In some embodiments, a LAMP reaction uses twoor three primer sets, and a polymerase with high strand displacementactivity in addition to a replication activity. (See Nagamine et al.,(2002) Mol. Cell. Probes 16 (3): 223-9; and U.S. Pat. No. 6,410,278,each of which is incorporated herein by reference).

LAMP was originally invented and formulated as an isothermalamplification with a requirement for four primers: two loop-generatingprimers (FIP and BIP comprising F1, F2 and B1, B2 priming sites,correspondingly) and two “Displacement primers” (F3 and B3). In order toincrease the speed of LAMP-based assays additional “Loop primers” wereadded in conjunction with the other primers used in LAMP, which resultedin significantly faster assays.

In some embodiments, LAMP amplification of a THCA synthase gene sequenceincludes contacting nucleic acid from a Cannabis plant with four or moreprimers that are at least 70%, 75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any ofSEQ ID NOs: 8-12.

In some embodiments, LAMP amplification of a CBDA synthase gene sequenceincludes contacting nucleic acid from a Cannabis plant with four, five,or six, or more primers that are at least 70%, 75%, 80%, 85%, 85%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to any of SEQ ID NOs: 13-18 and 27.

In some embodiments, LAMP amplification of a CBDA^(mut) (CBDA^(del))synthase gene sequence includes contacting nucleic acid from a Cannabisplant with four, five, or six, or more primers that are at least 70%,75%, 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 13, 15-19, and27.

In some embodiments, a LAMP amplification assay as described herein is acolorimetric LAMP assay.

Due to the specific nature of the action of these primers, the amount ofDNA produced in LAMP is considerably higher than PCR basedamplification. The reaction can be followed in real-time either bymeasuring the turbidity or by fluorescence using intercalating dyes. Dyemolecules intercalate or directly label the DNA, and in turn can becorrelated to the number of copies initially present. Hence, LAMP canalso be quantitative. Thus, LAMP provides major advantages due to itssimplicity, ruggedness, and low cost, and has the potential to be usedas a simple screening assay in the field or at the point of care byclinicians.

Primer design for LAMP assays generally involves selection of eightseparate regions of a target nucleic acid sequence (the FIP and BIPprimers encompass two primer binding sites each), with BIP/FIP and Loopprimers having significant restrictions on their positioning respectiveto each other. “Loop primers” are positioned between B2 and B1 sites andF2 and F1 sites, respectively, and must be orientated in a particulardirection. Further, significant care must be taken in primer design toavoid primer-dimers between the six primers (which can be especiallydifficult as the FIP and BIP primers are generally greater than 40nucleotides long). As a consequence, LAMP primer design is extremelychallenging, especially when targeting highly polymorphic markers andsequences containing complex secondary structure. At least in partbecause primer design for LAMP is subject to numerous constraints,software is generally used to assist with LAMP primer design.

LAMP has been observed to be less sensitive than PCR to inhibitors incomplex samples such as blood, likely due to use of a different DNApolymerase (typically Bst DNA polymerase rather than Taq polymerase asin PCR). LAMP is useful primarily as a diagnostic or detectiontechnique, but is generally not useful for some molecular biologyapplications enabled by PCR, such as, for example, cloning.

Also, multiplexing approaches for LAMP are relatively undeveloped. Thelarger number of primers per target in LAMP increases the likelihood ofprimer-primer interactions for multiplexed target sets. The product ofLAMP is a series of concatemers of the target region, giving rise to acharacteristic “ladder” or banding pattern on a gel, rather than asingle band as with PCR. Although this is not a problem when detectingsingle targets with LAMP, “traditional” (endpoint) multiplex PCRapplications wherein identity of a target is confirmed by size of a bandon a gel are not feasible with LAMP. Multiplexing in LAMP has beenachieved by choosing a target region with a restriction site, anddigesting prior to running on a gel, such that each product gives riseto a distinct size of fragment, although this approach adds complexityto the experimental design and protocol. The use of a strand-displacingDNA polymerase in LAMP also precludes the use of hydrolysis probes, e.g.TaqMan probes, which rely upon the 5′-3′ exonuclease activity of Taqpolymerase.

In some embodiments, a modified LAMP technique called LAMP-STEM is usedin a method of the present disclosure. LAMP-STEM system utilizes “Stemprimers,” which are directed to the stem portion of the LAMP amplicon(or “dumbbell”). Stem primers can be used as an alternative to LAMP“Loop primers.” When used in addition to loop-generating anddisplacement primers, Stem primers offer similar benefits in speed andsensitivity to the Loop primers. (See Gandelman et al., Loop-MediatedAmplification Accelerated by Stem Primers. Int. J. Mol. Sci. 2011,v12:9108-9124, and US 2012/0157326, each of which is incorporated hereinby reference). This beneficial effect of Stem primers is surprising asthey do not bind to the single-stranded DNA loops, which define the verynature of the LAMP technology. Stem primers can be employed in eitherorientation, do not require either the B2/B1 or F2/F1 sites to be aspecific distance apart, can be multiplexed, and allow the F1 and B1sites to be positioned further from each other than in LAMP.

Stem primers significantly accelerate LAMP comprised of loop-generatingand displacement primers only. They can be used on their own orsynergistically with other Stem primers or even Loop primers. Additionof Stem primers into LAMP has a positive effect on both speed andsensitivity. In some cases they improve reproducibility at low copynumber. The action of Stem primers can be rationalized via the proposedmechanism of LAMP. They anneal to transiently single-stranded regions ofthe amplicon and recopy the entire binding sites for the BIP/FIPprimers. An additional unique feature is the extra strongintra-molecular self-priming when Stem primers delimit amplicon.

In general, positioning of Stem primers is less constrained than that ofLoop primers. A rather challenging primer design involving selection ofat least eight binding sites is thus simplified. Furthermore, Stemprimers impose fewer limitations on the primer design in terms of stemlength, orientation and distances between B1-B2 and F1-F2 sites. Incontradiction to the postulated LAMP mechanism that relies on theinvolvement of displacement primers Stem primers can occasionally allowdisplacement primers not to be used at all, though it is not clear whythis is so. This has a major implication for primer design, as it allowsthe ability to omit one displacement primer or even both, if necessary.

qPCR

In some embodiments, an in vitro nucleic acid amplification assay foruse in the methods of the present disclosure is qPCR. Real-timequantitative polymerase chain reaction (qPCR) determines, the fractionalcycle number (C_(t)) at which the well's rising fluorescence(proportional to product formation) crosses a set threshold that isseveral standard deviations above the baseline fluorescence (Higuchi, etal., (1993) Kinetic PGR analysis: real-time monitoring of DNAamplification reactions, Biotechnology (NY), 11: 1026-1030, which isincorporated by reference in its entirety). The C_(t) versus log (amountof input target DNA) plot is linear, allowing relative quantitation ofunknowns by comparison to a standard curve derived from amplifying, inthe same plate, serial dilutions of a reference DNA sample.

In some embodiments, a qPCR assay is normalized, for example, a signalfrom a target sequence can be normalized to a signal from a referencesequence. In some embodiments, a target sequence and reference sequenceare measured in separate (monoplex) reactions. In some embodiments, areaction includes a dye that fluoresces upon intercalation into anydouble-stranded DNA, e.g., ethidium bromide or SYBR Green I, etc. Insome embodiments, a target sequence and reference sequence are measuredin the same reaction vessel via a multicolor multiplex qPCR. In someembodiments, a multiplex qPCR uses separate fluorescent dyes withdistinct excitation/emission spectra for each of the DNA sequences beingquantified (Wittwer, et al., (2001) Real-time multiplex PCR assays.Methods, 25, 430-442, which is incorporated by reference in itsentirety).

Kits

In some embodiments, the present disclosure provides kits comprisingmaterials useful for amplification and detection and/or sequencing ofCannabis plant nucleic acid (e.g., DNA). In some embodiments, Cannabisplant nucleic acid sample includes detection of all or part of a THCAsynthase gene sequence and/or a CBDA synthase gene sequence as describedherein. In some embodiments, a kit in accordance of the presentdisclosure is portable.

Suitable amplification reaction reagents that can be included in aninventive kit include, for example, one or more of: buffers; enzymeshaving polymerase activity; enzyme cofactors such as magnesium ormanganese; salts; nicotinamide adenide dinuclease (NAD); anddeoxynucleoside triphosphates (dNTPs) such as, for example,deoxyadenosine triphospate; deoxyguanosine triphosphate, deoxycytidinetriphosphate and deoxythymidine triphosphate, biotinylated dNTPs,suitable for carrying out the amplification reactions.

In some embodiments, a kit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more primer sequences for invitro nucleic acid amplification. Primer sequences may be suitable forin vitro nucleic acid amplification with any of the methods describedherein (e.g., QT-PCR, LAMP, etc.). In some embodiments, a kit of thepresent disclosure includes reagents suitable to perform a colorimetricLAMP assay for amplification of one or more Cannabis gene sequences asdescribed herein.

Depending on the procedure, a kit may further include one or more of:wash buffers and/or reagents, hybridization buffers and/or reagents,labeling buffers and/or reagents, and detection means. The buffersand/or reagents included in a kit are preferably optimized for theparticular amplification/detection technique for which a kit isintended. Protocols for using these buffers and reagents for performingdifferent steps of the procedure may also be included in a kit.

In some embodiments, a kit may further include one or more reagents forpreparation of nucleic acid from a plant sample. For example, a kit mayfurther include one or more of a lysis buffer, a DNA preparationsolution (e.g., a solution for extraction and/or purification of DNA).Kits may also contain reagents for the isolation of nucleic acids frombiological specimen prior to amplification. Protocols for using thesereagents for performing different steps of the procedure may also beincluded in a kit.

Furthermore, kits may be provided with an internal control as a check onthe amplification procedure and to prevent occurrence of false negativetest results due to failures in the amplification procedure. An optimalcontrol sequence is selected in such a way that it will not compete withthe target nucleic acid sequence in the amplification reaction (asdescribed above).

In some embodiments, a kit may further include reagents for anamplification assay to characterize the gender of a Cannabis plant.

Reagents may be supplied in a solid (e.g., lyophilized) or liquid form.Kits of the present disclosure may optionally comprise differentcontainers (e.g., vial, ampoule, test tube, flask or bottle) for eachindividual buffer and/or reagent. In some embodiments, each componentwill generally be suitable as aliquoted in its respective container orprovided in a concentrated form. Other containers suitable forconducting certain steps of inventive amplification/detection assay(s)may also be provided. Individual containers of a kit are preferablymaintained in close confinement for commercial sale.

A kit may also comprise instructions for using the amplificationreaction reagents, primer sets, primer/probe sets according to thepresent disclosure. Instructions for using a kit according to one ormore methods of the present disclosure may comprise instructions forprocessing the biological sample, extracting nucleic acid molecules,and/or performing one or more amplification reactions; and/orinstructions for interpreting results. In some embodiments, a kit maycomprise instruction for determining, assessing and/or classifying aCannabis plant used in the described methods as a Type Ia, Type Ib, TypeIIa, Type IIb, Type IIc, Type IIIa, Type IIIb, Type IV or Type V plant.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXEMPLIFICATION Example 1: Genotyping Cannabis Plants

Bt and Bd alleles have been proposed as a model for generating Type I,II, III, and IV plants. While this classification system can bepredictive, the exact DNA sequences that govern them remain unknown.Single molecule sequencing was performed to identify mutations in genesequences in cannabinoid synthesis pathway(s) (e.g., THCA synthase andCBDA synthase). This sequencing revealed a more refined structure of theinheritance and revealed numerous additional subtypes that can beidentified by genotyping to predict chemical inheritance. See, FIG. 2.As illustrated in FIG. 2, a Cannabis plant may be characterized as aType Ia, Type Ib, Type IIa, Type IIb, Type IIc, Type IIIa, Type IIIb,Type IV or Type V plant.

Example 2: Assay for Genotyping THCA Synthase

This example describes development of superior isothermal nucleic acidamplification assays for detection of THCA synthase. Specifically, acolorimetric LAMP assay for detection of THCA synthase is described. Itis envisioned that such a colorimetric LAMP assay may be portable, sothat can it be performed at a point of grow to resolve Cannabischemotypes in a rapid and scalable manner.

Challenges with genotyping a THCA synthase include, for example, (1)that there are many pseudogene sequence copies of THCA synthase and (2)that THCA synthase gene sequences are highly polymorphic. For at leastthis reason, designing primers can be difficult and testing such primerson a few samples does not provide adequate population diversity toguarantee assay performance in the broader markets. The presentdisclosure addresses these deficiencies with a novel Long Range (2130bp) LAMP assay that includes a primer that anneals to a promoter regionof THCA synthase and internal primers that are less prone to effectsresulting from polymorphisms.

Weiblen, Stagginus and Onofri suggested primers for THCA synthasedetection but failed to test enough samples to find fatal polymorphismsin their detection approach (Weiblen et al., (2015), The NewPhytologist, PMID: 26189495; Staginnus et al., (2014) Journal ofForensic Sciences, 59(4):919-26, PMID: 24579739; and Onofri et al.,(2015), supra, each of which is incorporated by reference in itsentirety). While Kitamura et al. described a portable LAMP assay forTHCA synthase, the assay was performed using only two THCA+ cultivarsand one fiber type. Consequently, Kitamura et al. did not consider thefull complexity of cannabis genotypes in circulation. Moreover, theprimers used in Kitamura's assay annealed to different regions in a THCAsynthase gene sequence. As a result, and shown herein, the presentdisclosure recognizes that the Kitamura primers were insufficient fordetecting/amplifying a THCA synthase gene sequence in numerous commonCannabis cultivars. In fact, sequence analysis of the Kitamura primersrevealed that the 5′ end of the Kitamura BIP primer sits on a commonI63E and I63F polymorphisms described by Onofri et al., (2015), supra,which is incorporated by reference in its entirety. As this 5′ endbecomes the 3′ end in the second strain synthesis of a LAMP reaction,presence of this polymorphism could impair or stall the LAMP reaction. Asimilar problem existed with other Kitamura primers, which traversed theP333R variant and the Glu265Gln variants described by Onofri, et al.,(2015), supra, incorporated by reference in its entirety.

Amplification issues resulting from polymorphisms in an underlying genesequence is a common problem with genetic studies of THCA synthase genesequences. THCA synthase gene sequences are under selective breedingpressure and has a 4-fold higher polymorphism rate than the rest of thegenome. There is a SNP approximately every 25 nucleotides in THCAsynthase gene sequences, making primer design internal to a THCAsynthase gene sequence complicated. To address this, primers (e.g., F3and B3 primers for LAMP) were designed that were external to a THCAsynthase gene sequence, where there is more sequence conservation. Thisresults in a THCA synthase gene sequence amplicon of greater than 2000nucleotides. There was no expectation that such a reaction would work,as there were no reports of Long Range LAMP assays in the literature andthe common design tools found at Primer Explorer did not allow primerdesign of targets this large.

Materials and Methods

LAMP assay

-   -   10 ul 2×LAMP mix (NEB Catalog #M1800S)    -   2 ul of primer cocktail    -   6 ul ddH20 (pH 6.5)    -   2 ul DNA (4 mm leaf biopsy boil in 100 ul ddH20, 5% Chelex)    -   Exemplary THCA synthase Long Range LAMP primers are provided in        FIG. 9 and Table 2:

TABLE 2 THC_BIP CACACAAGCACGTATTTGGGCTT SEQ ID NO: 8TAGTTTTCACCTTAACTAACCT THCFTP GACTCGCATGATTAGTTTTTCCT SEQ ID NO: 9ATCCTTATGTGTCCCAAAATCC THC_Loop1 TCCCTATAATTGAGATACGCCAAT SEQ ID NO: 10THC-F3 ATGGGAACCATAATAAACTATAA SEQ ID NO: 11 AAGTCATT THC_B3TATGATTGTCTACACAGTTCTAA SEQ lD NO: 12 TGTAGATTTTC

To compare efficacy of Kitamura primers (“K primers”) to exemplary LongRange primers (“LR primers”), LAMP assays were performed with theseprimers on various strains of Cannabis plants. Results are shown in FIG.5, panel (A). In FIG. 5, panel (A), the text on the left side of theimage indicates the strain of Cannabis plant for each row of samplesthat was tested by LAMP amplification. The top row included samples fromGranddaddy Purple (GDP), the second row from the top included samplesfrom Otto, the third row from the top included samples from a first hempstrain, the fourth row from the top included samples from a second hempstrain, the fifth row from the top included samples from Grape Stomper(RSP10516), and the bottom row included samples from Erk Train XDeadhead OG (RSP10517). A sample obtained from each strain was placed inthe first and third columns. For each strain, a LAMP assay using the Kprimers was performed in the first column, and a LAMP assay using the LRprimers was performed in the third column.

As shown in FIG. 5, panel (A), the two hemp strains displayed a negativeresult for THCA synthase using either the K primers or the LR primers.As a hemp strain would be expected to be THCA⁻/THCA⁻, this result showedthat LAMP amplification using either the K primers or the LR primers wasnot giving a false positive result. The Otto strain, which expressesTHCA synthase, displayed a positive result using the K primers, whichshowed that LAMP amplification using the K primers detected the presenceof a THCA synthase gene. However, the GDP strain of Cannabis plant,which also expresses THCA synthase, had a negative result when LAMPamplification was performed with K primers, while giving a positiveresult when LAMP amplification was performed with LR primers. Thisresults showed that LR primers are able to successfully detect a THCAgene sequence by LAMP amplification, and do so in strains for which Kprimers are unable to detect a THCA gene sequence by LAMP amplification.

To determine how a Long Range LAMP assay of THCA synthase would performat various temperatures, LAMP assays with LR primers were performed onvarious samples over a range of temperatures (temperatures indicated onright side of FIG. 5, panel (B)). As indicated in FIG. 5, panel (B), thefirst two columns of samples were obtained from THCA synthase negativehemp strains. The third column included samples from a THCA synthasepositive Cannabis strain. The fourth column included samples from aGranddaddy purple strain. The eighth column included a negative control,and the ninth column included a positive control. Amplification of theTHCA synthase gene sequence was successful for the THCA synthasepositive Cannabis strain, the Granddaddy Purple strain, and the positivecontrol at each of the temperatures tested. These data showed that LAMPassays using LR primers were able to properly detect THCA synthasepositive strains across a broad temperature profile.

The exemplary Long Range LAMP assay of THCA synthase was repeated onvarious different Cannabis strains that are known to be THCA synthasepositive. See, Figure. 6. FIG. 6, depicts LAMP amplifications with LRprimers on 28 different known THCA synthase positive strains of Cannabisat 65° C., for 50 minutes. Wells H6-H9 were control samples. As can beseen, all 28 THCA synthase positive samples had successful amplificationwith the exemplary LR primers.

Accordingly, this example demonstrates that LAMP assays using long rangeprimers (such as the exemplified LR primers) of THCA synthase genesequences was robust and reliable. This example also demonstrates theshortcomings of LAMP amplification using the Kitamura primers foraccurately detecting THCA synthase gene sequences in Cannabis strains.

Example 3: Combined THCA Synthase/CBDA Synthase Assay

This example describes characterization of the CBDA synthase Bd alleleas a four nucleotide deletion within the coding sequence of this gene.This example also describes development of amplification assays fordetection of wild-type (CBDA^(wt)) and mutant (CBDA^(Bd)) CBDA synthasegene sequences.

Kitamura simply assessed presence of a single gene sequence (THCAsynthase) and its deleted pseudogene sequence copies in certain hempfiber varietals. Thus, the LAMP assay described in Kitamura (at best)only differentiated Type I or II Cannabis from other types (Type II-V),but failed to refine certain chemotype categories. To more rigorouslystratify the numerous classes of cannabinoid production other genesequence(s) that compete for the same cannabinoid precursor (e.g.,cannabigerolic acid or CBGA) should be considered. Since both THCAsynthase and CBDA synthase compete for cannabigerolic acid, themutational spectrum of both of these gene sequences with respect to oneanother was examined.

To characterize CBDA synthase, single molecule sequencing of CBDAsynthase in dozens of Cannabis cultivars was performed. This sequencingrevealed a 4 nucleotide frame shifting deletion that governs the Bdallele. The Bd allele was then surveyed in more than 50 hemp and CBDlines to confirm the Bd allele and its phenotype. A consensus sequencefor the region of the CBDA synthase gene sequence that includes thisdeletion is shown in FIG. 4.

Assays that targeted a 4 nucleotide deletion in the CBDA synthase genesequence (4bpDel) (See, FIG. 4, residues 330-333) were designed. Variousdifferent hemp lines that were known to be THCA synthase negative weretested by both an exemplary CBDA synthase assay and an exemplary LAMPassay using LR primers for a THCA synthase gene sequence. FIG. 7 showsLAMP assays for amplification of wild-type CBDA synthase gene sequence(panel (A)) and THCA synthase gene sequence (panel (B)) on 38 differenthemp strains (i.e., THCA synthase negative strains). A plate mapdescribing the hemp strain in each well is provided at the bottom (panel(C). As shown in FIG. 7, panel (A), all samples but one (sample M2 inwell A1) resulted in amplification of a wild-type CBDA synthase genesequence. Further, as shown in FIG. 7, panel (B), LAMP assay using LRprimers for a THCA synthase gene sequence consistently showed noamplification on hemp samples. These results showed that the chemotypeof Cannabis strains were accurately detected by performance of a CBDAsynthase assay and an LAMP amplification using LR primers for a THCAsynthase gene sequence.

For the one hemp sample (A1) that failed to amplify in the exemplaryCBDA synthase gene sequence assay, when this sample was repeated using adifferent DNA purification method, amplification of CBDA synthase genesequence was observed. Thus, it was concluded that the result for thisparticular sample was a false negative that resulted from otherexperimental conditions.

With two assays, discernment of THCA synthase positive plants from CBDAsynthase positive plants was achieved. The copy number of THCA synthaseor CBDA synthase in the genome of Cannabis plants could not be detected,however. Copy number can be useful information for breeders looking tobreed plants that produce high levels of THC or CBD. In order to assesscopy number, a deletion in the CBDA synthase gene sequence was targeted.

Exemplary CBDA^(wt) synthase primers (which hybridize to a wild-typeCBDA synthase gene sequence) are provided in FIG. 9 and Table 3:

TABLE 3 CBD_BIP CAATCTTAGATTCACCTCTGAC SEQ ID NO: 13ACAAGACATGTGAAGGAGTGAC CBD_FlP TGTATTGTCGAATTTAGGACAGAC SEQ ID NO: 14AATGCAACAAATCTAAAACTCGTA CBD_Loop1 CAATGGGTTGTTTTGAGTGT SEQ ID NO: 15CBD_Loop2 ACCCCAAAACCACTTGT SEQ ID NO: 16 CBD_F3 CTTCTCGCAATATATTCCCAATSEQ ID NO: 17 CBD_F3_v2 ATGCTTCTCGCAATATATTCCCA SEQ ID NO: 27 CBD_B3GCATAGAATAGTGCCTTGGAT SEQ ID NO: 18

Exemplary CBDA^(del) synthase primers (which hybridize to a mutant CBDAsynthase gene sequence including a 4 nucleotide deletion) are providedin FIG. 9 and Table 4:

TABLE 4 CBD_BIP CAATCTTAGATTCACCTCTGAC SEQ ID NO: 13ACAAGACATGTGAAGGAGTGAC CBD-NEG_FIP TGTATTGTCGAATTTAGGACAGACSEQ ID NO: 19 AATGCAACAAATCTAAAACTTACA CBD_Loop1 CAATGGGTTGTTTTGAGTGTSEQ ID NO: 15 CBD_Loop2 ACCCCAAAACCACTTGT SEQ ID NO: 16 CBD_F3CTTCTCGCAATATATTCCCAAT SEQ ID NO: 17 CBD_F3_v2 ATGCTTCTCGCAATATATTCCCASEQ ID NO: 27 CBD_B3 GCATAGAATAGTGCCTTGGAT SEQ ID NO: 18

The present disclosure encompasses a recognition that detection of bothwild-type and mutant CBDA gene sequences will enable geneticdifferentiation of strains, for example, to differentiate a CBDA⁻/CBDA⁺genotype from a CBDA⁺/CBDA⁺ genotype.

FIG. 8 depicts results obtained from exemplary LAMP assays foramplification of CBDA^(wt) and CBDA^(del) (e.g., CBDA^(Bd)) genesequences. FIG. 8, panel (A), depicts results obtained from LAMP assayswith CBDA^(wt) primers on various hemp samples. Most of the samplesresulted in amplification of a CBDA^(wt) gene sequence. FIG. 8, panel(B), depicts results from LAMP assays with CBDA^(del) primers on varioushemp samples. As can be seen, most of these samples did not amplify aCBDA^(del) gene sequence.

The present disclosure encompasses a recognition that detection of bothwild-type and mutant THCA gene sequences will enable geneticdifferentiation of strains. In order to assess THCA synthase copynumber, one or more sequence variants can be targeted. For example,primers can be used that target the Cannabis THCA synthase A411Vvariant.

Thus, combined THCA synthase/CBDA synthase assays provided herein candifferentiate additional types of Cannabis plants. In some embodiments,methods in the context of the present disclosure can differentiate TypeI, Type II, Type III, Type IV, and Type V plants. As described herein,the present disclosure further provides new sub-classification of plantswe term Type Ia, Type Ib, Type IIa, Type IIb, Type IIc, Type IIIa, TypeIIIb, Type IV and Type V Cannabis plants, see, e.g., FIG. 2 and Table 1(above).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of an invention described herein. The scope of an inventiondescribed is not intended to be limited to the above Description, butrather is as set forth in the following claims:

What is claimed is:
 1. A method of detecting whether atetrahydrocannabinolic acid (THCA) synthase gene sequence and acannabidiolic acid (CBDA) synthase gene sequence are present in aCannabis plant, comprising: obtaining a sample from the Cannabis plantthat contains nucleic acids; contacting the nucleic acids with primersspecific for the THCA synthase gene sequence and primers specific forthe CBDA synthase gene sequence; amplifying the THCA synthase genesequence and/or CBDA synthase gene sequence when present among thenucleic acids; and detecting amplicons upon amplification of the THCAsynthase gene sequence and/or CBDA synthase gene sequence, wherein: theprimers specific for the THCA synthase gene sequence include a primerspecific for a promoter sequence of the THCA synthase gene sequence; theprimers specific for the CBDA synthase gene sequence include a primerspecific for presence or absence of SEQ ID NO: 3 so as to indicate thepresence or absence of SEQ ID NO: 3 upon amplification; andamplification of the THCA synthase gene sequence indicates that the THCAsynthase gene sequence is at least 2000 nucleotides long.
 2. The methodof claim 1, wherein the primer specific for the promoter sequence of theTHCA synthase gene sequence binds to the promoter sequence at a locationthat is 200-1000 nucleotides upstream of a THCA synthase open readingframe.
 3. The method of claim 1, wherein the THCA synthase gene sequenceis between 2100-2200 nucleotides long.
 4. The method of claim 1, whereinthe CBDA synthase gene sequence is a wild-type CBDA synthase genesequence that includes SEQ ID NO:
 3. 5. The method of claim 1, whereinthe CBDA synthase gene sequence is a mutant CBDA synthase gene sequencehaving a deletion of SEQ ID NO:
 3. 6. The method of claim 1, wherein theCannabis plant is a C. sativa, C. indica, or C. ruderalis plant.
 7. Themethod of claim 1, wherein the Cannabis plant is characterized as a TypeIa, Type Ib, Type IIa, Type IIb, Type IIc, Type IIIa, Type IIIb, TypeIV, or Type V plant.
 8. The method of claim 1, wherein the sampleincludes sample portions and the nucleic acids of a first sample portionare contacted with the primers specific for the THCA synthase genesequence and the nucleic acids of a second sample portion are contactedwith the primers specific for the CBDA synthase gene sequence.
 9. Themethod of claim 1, wherein a polymerase chain reaction (PCR) isperformed.
 10. The method of claim 1, wherein an isothermal nucleic acidamplification is performed.
 11. The method of claim 1, wherein aLoop-Mediated Isothermal Amplification (LAMP) assay is performed. 12.The method of claim 1, wherein a colorimetric assay is performed. 13.The method of claim 1, wherein the primers specific for the THCAsynthase gene sequence include at least five primers.
 14. The method ofclaim 1, wherein the primers specific for the CBDA synthase genesequence include at least five primers.
 15. The method of claim 1,wherein the primers specific for the THCA synthase gene sequence includea primer comprising a sequence complementary to 20-60 nucleotides of SEQID NO: 6 and/or a primer comprising a sequence complementary to 20-60nucleotides of SEQ ID NO:
 7. 16. The method of claim 1, wherein theprimers specific for the THCA synthase gene sequence comprise at leastone primer comprising a sequence that is at least 70% identical to anyof SEQ ID NOS: 8-12.
 17. The method of claim 1, wherein the primersspecific for the THCA synthase gene sequence comprise at least oneprimer comprising a sequence that is at least 80% identical to any ofSEQ ID NOS: 8-12.
 18. The method of claim 1, wherein the primersspecific for the THCA synthase gene sequence comprise at least oneprimer comprising a sequence that is at least 80% identical to SEQ IDNO: 11 or SEQ ID NO:
 12. 19. The method of claim 1, wherein the primersspecific for the THCA synthase gene sequence comprise at least oneprimer comprising a sequence that is at least 90% identical to any ofSEQ ID NOS: 8-12.
 20. The method of claim 1, wherein the primersspecific for the CBDA synthase gene sequence comprise at least oneprimer comprising a sequence that is at least 70% identical to any ofSEQ ID NOS: 13-19 and
 27. 21. The method of claim 1, wherein the primersspecific for the CBDA synthase gene sequence comprise at least oneprimer comprising a sequence that is at least 80% identical to any ofSEQ ID NOS: 13-19 and
 27. 22. The method of claim 1, wherein the primersspecific for the CBDA synthase gene sequence comprise at least oneprimer comprising a sequence that is at least 80% identical to SEQ IDNO: 14 or SEQ ID NO:
 19. 23. The method of claim 1, wherein the primersspecific for the CBDA synthase gene sequence comprise at least oneprimer comprising a sequence that is at least 90% identical to any ofSEQ ID NOS: 13-18 and
 27. 24. The method of claim 1, wherein the primersspecific for the CBDA synthase gene sequence comprise at least oneprimer comprising a sequence that is at least 90% identical to any ofSEQ ID NOS: 13, 15-19, and
 27. 25. A method of detecting whether atetrahydrocannabinolic acid (THCA) synthase gene sequence and acannabidiolic acid (CBDA) synthase gene sequence are present in aCannabis plant, comprising: obtaining a sample from the Cannabis plantthat contains nucleic acids; contacting the nucleic acids with primersspecific for the THCA synthase gene sequence and primers specific forthe CBDA synthase gene sequence; amplifying the THCA synthase genesequence and/or CBDA synthase gene sequence when present among thenucleic acids; and detecting amplicons upon amplification of the THCAsynthase gene sequence and/or CBDA synthase gene sequence, wherein: theprimers specific for the THCA synthase gene sequence include a primerspecific for a promoter sequence of the THCA synthase gene sequence; theprimers specific for the CBDA synthase gene sequence include a primerspecific for presence or absence of SEQ ID NO: 3 so as to indicate thepresence or absence of SEQ ID NO: 3 upon amplification; and the primerspecific for the promoter sequence of the THCA synthase gene sequencebinds to the promoter sequence at a location that is 200-1000nucleotides upstream of a THCA synthase open reading frame.
 26. Themethod of claim 25, wherein the primers specific for the THCA synthasegene sequence further include a primer that binds to a sequence that is50-1000 nucleotides downstream of the THCA synthase open reading frame.27. A method of detecting whether a tetrahydrocannabinolic acid (THCA)synthase gene sequence and a cannabidiolic acid (CBDA) synthase genesequence are present in a Cannabis plant, comprising: obtaining a samplefrom the Cannabis plant that contains nucleic acids; contacting thenucleic acids with primers specific for the THCA synthase gene sequenceand primers specific for the CBDA synthase gene sequence; amplifying theTHCA synthase gene sequence and/or CBDA synthase gene sequence whenpresent among the nucleic acids; and detecting amplicons uponamplification of the THCA synthase gene sequence and/or CBDA synthasegene sequence, wherein: the primers specific for the THCA synthase genesequence include a primer specific for a promoter sequence of the THCAsynthase gene sequence; the primers specific for the CBDA synthase genesequence include a primer specific for presence or absence of SEQ ID NO:3 so as to indicate the presence or absence of SEQ ID NO: 3 uponamplification; and the amplicons of the THCA synthase gene sequenceinclude at least part of the THCA synthase promoter and 5′ UTR.